EXCHANGE 


The  Influence  of  Calcium,   Magnesium 

and  Potassium  Nitrates  upon  the 

Toxicity    of  Certain    Heavy 

Metals  Toward  Fungus 

Spores 


DISSERTATION 

Submitted  to  the  Board  of  University  Studies  of  the  Johns 
Hopkins  University  in  conformity  with  the  require- 
ments for  the  degree  of  Doctor  of  Philosophy 


By 
LON  A.  HAWKINS 


Baltimore,  June,  1913 

[.PHYSIOLOGICAL    RESEARCHES    VOL.     I,    NO.    2,    AUGUST,     1013] 


The  Influence  of  Calcium,   Magnesium 

and  Potassium  Nitrates  upon  the 

Toxicity    of  Certain    Heavy 

Metals  Toward  Fungus 

Spores 


Submitted  to  the  Board  of  University  Studies  of  the  Johns 
Hopkins  University  in  conformity  with  the  require- 
ments for  the  degree  of  Doctor  of  Philosophy 


By 
LON  A.  HAWKINS 


-*-?•-.- 


Baltimore,  June,  191. '5 

[PHYSIOI.nGirAJ.    RKKKARrllKS    VOt.     I,    NO.     2,    AtTl-.VST, 


BIO'-OGY 
RA 
G 


THE    INFLUENCE    OF    CALCIUM,    MAGNESIUM    AND 

POTASSIUM  NITRATES  UPON  THE  TOXICITY 

OF  CERTAIN  HEAVY  METALS  TOWARD 

FUNGUS  SPORES.1 

LON   A.   HAWKINS. 

ABSTRACT.2 

This  study  has  to  do  with  the  influence  of  one  salt  in  altering  the  toxic 
effect  of  another  upon  fungus  spores.  It  is  shown  that  the  toxic  effect 
of  copper  nitrate  on  the  germination  of  Gloeosporium  spores  can  be  in- 
hibited or  modified  by  the  addition  to  the  medium  of  calcium  nitrate  and 
that  the  molecular  ratio  of  the  quantity  of  the  calcium  salt  thus  required 
to  the  amount  of  copper  nitrate  present  increases  with  the  concentration 
of  the  latter.  This  effect  is  apparently  the  result  of  a  simultaneous  action 
of  the  two  salts  upon  the  organism  and  cannot,  in  the  cases  here  con- 
sidered, be  related  either  to  formation  of  an  undissociated  double  salt  or 
to  depression  of  the  ionization  of  the  toxic  salt  because  of  the  ion  common 
to  the  two  salts.  Potassium  nitrate  is  also  effective  in  inhibiting  or 
modifying  the  toxicity  of  copper  nitrate.  The  influence  of  calcium  upon 
the  toxicity  of  copper  is  of  interest  in  connection  with  the  problem  of 
fungicides  and  fungicidal  action. 

The  toxicity  of  lead  nitrate  is  similarly  influenced  by  the  presence  of 
calcium  nitrate,  and  the  molecular  proportions  in  which  these  two  salts 
may  be  combined  so  as  just  to  counteract  the  toxicity  of  the  former  were 
found  to  be  the  same  for  the  three  different  concentrations  of  lead 
nitrate  that  were  employed.  The  toxicity  of  this  lead  salt  is  likewise  influ- 
enced by  proper  concentrations  of  magnesium  nitrate.  Both  calcium  nitrate 
and  magnesium  nitrate  markedly  decrease  the  toxicity  of  zinc  nitrate,  but 
neither  exhibits  any  effect  on  the  toxicity  of  aluminum  nitrate  in  the  con- 
centrations used  in  these  experiments. 

The  effects  produced  by  the  various  single  salts  upon  the  germinating 
spores  are  of  interest.  Four  types  of  response  to  the  salt  solution  are 
clearly  discernible,  (i)  The  effect  of  the  solution  may  be  the  same  as 
that  of  distilled  water,  the  spores  germinating  normally,  with  long,  narrow 
tubes.  (2)  A  somewhat  higher  concentration  of  the  toxic  substance  may 

'Botanical  contribution  from  the  Johns  Hopkins  University  No.   31. 

2  The  manuscript  of  this  paper  was  received  April  i,  1913.  This  abstract  was  preprinted,  without 
change,  from  these  types  and  was  issued  as  Physiological  Researches  Preliminary  Abstracts  vol.  i,  no. 
2,  August,  1913. 

57 

PHYSIOLOGIC/ L  RESEARCHES    VOL.   I,  NO.  2^  SERIAL  NO.   2, 
AUGUST, 


"814805 


58  LON  A.  HAWKINS 

allow  germination,  this  being  abnormal,  however,  with  the  production  of 
unusual  and  characteristic  structures.  (3)  Germination  may  be  inhibited 
for  eighteen  hours,  the  spores  germinating  when  subsequently  transferred 
from  the  toxic  solution  to  distilled  water.  (4)  The  spores  may  be  killed 
in  eighteen  hours  and  thus  be  incapable  of  renewed  activity  even  when 
transferred  to  water.  It  was  usually  possible  to  bring  about  these  four 
types  of  response  by  means  of  any  one  of  the  compounds  used  in  this  study 
by  a  proper  choice  of  concentration.  An  arrangement  of  these  substances 
in  the  order  of  their  toxicity,  as  evidenced  by  the  inhibition  of  spore 
germination,  is  found  to  hold  also  for  their  effectiveness  in  bringing  about 
the  other  and  less  final  changes  which  lead  to  abnormal  growth.  A  list 
of  the  substances  employed,  arranged  in  the  order  of  their  toxicity  follows : 
Cu(N03)2,  CuS04,  Pb(N03)2,  A1(N03)3,  HNO3,  Zn(NO3)2,  Ni(NO3)2, 
Mg(NO3)2,  Ca(NO3)2,  KNO3,  sucrose. 

It  is  shown  that  no  one  of  the  several  hypotheses  heretofore  proposed  to 
account  for  the  dynamics  of  the  antagonistic  salt  action  is  adequate  to 
explain  all  the  facts  brought  out  in  this  study. 

INTRODUCTION. 

Numerous  instances  have  been  recorded  of  the  influence  of  salts  on  the 
toxicity  exerted  by  various  substances  upon  organisms.  This  antagonistic 
action,  as  it  is  frequently  called,  of  a  salt  upon  a  toxic  substance,  is  of 
considerable  importance  in  influencing  the  behavior  of  organisms  in  a 
given  environment,  or  indeed  in  determining  whether  or  not  they  may 
exist  at  all  in  certain  environments.  For  example,  Loew3  has  shown  that 
the  toxic  effect  on  Spirogyra  of  a  one  per  cent,  solution  of  magnesium 
nitrate  is  inhibited  by  the  presence  in  the  medium  of  a  0.3  per  cent,  solu- 
tion of  calcium  nitrate,  while  Loeb4  has  demonstrated  that  the  addition  of 
a  small  quantity  of  a  calcium  salt  to  a  0.625111  solution  of  NaCl  inhibits 
the  toxic  effect  of  the  NaCl  on  the  development  of  Fundulus  eggs.  Oster- 
hout5  has  shown  that  a  physiologically  balanced  solution,  containing  sodium 
chloride,  magnesium  chloride,  magnesium  sulphate,  potassium  chloride  and 
calcium  chloride,  is  necessary  for  the  best  growth  of  certain  marine  algae. 
True  and  Bartlett6  have  brought  out  the  fact  that  a  ratio  of  one  molecule 
of  calcium  to  nine  molecules  of  magnesium  inhibits  the  toxic  effect  of 
rather  high  concentrations  of  magnesium  upon  roots  of  Canada  field 

3  Loew,  O.,  Ueber  die  physiologischen  Functionen  der  Calcium-  und  Magnesium-salze  im  Pflanzen- 
organismus.     Flora  75:  368-394.     1892. 

4  Loeb,  J.,  Ueber  den  Einflus  der  Werthigkeit,  und   moglicherweise  der  elektrischen   Ladung  von 
lonen  auf  ihre  antitoxische  Wirkung.     Archiv  ges.  Physiol.  88:  68-78.     1902. 

5  Osterhout,  W.  J.  V.,  On  the  importance  of  physiologically  balanced  solutions  for  plants.     I.  Marine 
plants.     Bot.  Gaz.  42:  127-134.     1906. 

'True,  R.  H.,  and  Bartlett,  H.H.,  Absorption  and  excretion  of  salts  as  influenced  by  concentration 
and  composition  of  culture  solutions.     U.S.  Dept.  Agric.  Bur.  Plant.  Ind.  Bulletin  231.     1912. 


TOXICITY  OF  HEAVY  METALS  59 

peas.  Other  cases  of  antagonistic  salt  action  in  combinations  of  salts 
of  the  alkali  metals  or  of  the  alkaline  earths  have  been  demonstrated, 
and  some  information  has  been  obtained  regarding  the  influence  of 
these  salts  on  the  effect  of  the  heavy  metals,  which  seem  to  be 
universally  toxic.  The  latter  have  not  received  much  attention,  however, 
and  it  seemed  worth  while  to  investigate  some  of  these,  alone  and  in  the 
presence  of  calcium  and  magnesium,  to  obtain  evidence  as  to  whether  the 
lighter  metals  may  modify  in  any  way  the  toxicity  of  the  heavy  ones. 
The  investigation  described  in  this  paper  was  accordingly  undertaken. 
The  problem  here  involved  will  be  taken  up  more  in  detail  after  some  of 
the  literature  pertinent  to  the  subject  has  been  considered. 

One  of  the  earlier  studies  of  the  influence  of  chemical  compounds  upon 
the  toxicity  of  the  heavy  metals  was  carried  out  by  Kronig  and  Paul.7 
These  authors  determined  the  effects  of  various  heavy  metals  in  com- 
bination with  many  salts  as  well  as  with  certain  acids  and  bases.  Mainly 
from  work  with  Bacillus  anthracis,  they  regard  the  influence  of  other 
halogen  compounds  upon  the  toxicity  of  mercuric  chloride  as  probably 
due  to  a  depression  of  the  ionization  of  the  latter  salt.  In  this  connection 
they  say  (page  no):  "  Die  Desinfectionswirkung  wasseriger  Mercuri- 
chloridlosungen  werden  durch  Zusatz  von  Halogenverbindungen  der  Metalle 
und  von  Salzaure  herabgesetzt.  Es  ist  wahrscheinlich,  dass  diese  Vermin- 
derung  der  Desinfectionskraft  auf  einer  Riickdrangung  der  elektrolytischen 
Dissociation  beruht." 

Clark8  carefully  studied  the  influence  of  various  concentrations  of 
sodium  chloride  upon  the  toxicity  of  mercuric  chloride  as  regards  the 
process  of  germination  in  various  fungus  spores  and  seeds  and  that  of 
growth  in  yeasts  and  bacteria.  He  found  that  the  toxicity  of  a  mercuric 
chloride  solution  increased  with  addition  of  small  quantities  of  sodium 
chloride  but  decreased  when  high  concentrations  of  the  sodium  salt 
were  used.  He  explained  these  phenomena  by  considering  that  a  double 
salt  of  sodium  chloride  and  mercuric  chloride  was  formed,  such  as 
Na2HgCl4  or  some  similar  combination.  He  supposed  that  the  dissociation 
tension  of  this  double  salt  was  probably  much  higher  than  that  of  mercuric 
chloride,  a  consideration  which  might  account  for  the  increased  toxicity 
of  combinations  in  which  small  amounts  of  sodium  chloride  were  employed. 
In  this  connection,  he  suggested  that  the  HgCl4  ion  present  when  such  a 
salt  as  Na2HgCl4  dissociated  at  lower  concentrations,  might  be  considerably 
more  toxic  than  the  HgCl2  molecule  to  which  he  seems  to  attribute  the 
toxicity  of  mercuric  chloride  solution. 

7  Kronig,  B.,  and  Paul,  Th.,  Die  chemischen  Grundlagen  der  Lehre  von  der  Giftwirkung  und  Desin- 
fection.     Zeitsch.  Hygiene  und  Infectionskrankheiten  25:   1-112.      1897. 

8  Clark,  J.  P.,  On  the  toxic  value  of  mercuric  chloride  and  its  double  salts.     Jour.  Physic.  Chem. 
5:  289-316.     1901. 


60  LON  A.  HAWKINS 

In  a  later  investigation,  on  the  toxicity  of  copper  in  combination  with 
various  chemical  compounds,  the  same  writer9  has  shown,  for  spore  germ- 
ination in  Oedoccphalum  albidum  and  Rhizopus  nigricans  that  ammonium 
nitrate,  sodium  sulphate,  potassium  sulphate,  and  potassium  chloride  all 
markedly  decreased  the  toxic  effects  of  both  copper  chloride  and  copper 
sulphate.  He  used  relatively  high  concentrations,  in  some  cases  five  per 
cent.,  of  the  alkali  and  ammonium  salts.  He  considers  the  decreased  toxicity 
just  mentioned  as  probably  due  to  the  formation  of  double  salts,  as  he 
suggested  in  the  case  of  mercuric  chloride. 

Le  Renard10  studied  the  comparative  toxicities  of  salts  of  many  of  the 
heavy  metals  upon  Penicillium,  the  fungus  being  grown  in  various  concen- 
trations of  nutrient  media.  He  used  the  acetates  of  potassium,  magnesium 
and  ammonium  alone,  and  the  acetates,  formates,  nitrates,  phosphates,  and 
sulphates  of  these  three  metals  with  glucose.  Also,  in  combination  with 
various  concentrations  of  the  salts  in  the  nutrient  medium  he  used  several 
concentrations  of  the  chlorides  and  nitrates  of  zinc,  nickel,  cobalt  and 
copper,  mercuric  chloride,  silver  nitrate,  and  the  sulphate  and  acetate  of 
copper.  The  presence  in  the  nutrient  medium  of  the  lighter  metals  in  higher 
concentration  was  usually  found  to  decrease  the  toxicity  of  the  heavy 
metals. 

True  and  Gies11  showed  that  calcium  modified  the  toxicity  of  various 
copper  salts,  of  zinc  sulphate  and  of  mercuric  chloride,  in  their  effect 
upon  the  growth  of  roots  of  Lupinus  albus.  In  discussing  their  results 
these  authors  say :  "  The  stimulating  action  of  the  calcium  seems  to  have 
operated  against  the  retarding  action  of  the  copper,  and  the  result  is  a 
marked  diminution  in  the  poisonous  action  of  the  copper."  They  thus 
relate  this  influence  of  calcium  upon  copper  to  a  mutual  effect  of  the  two 
salts  on  the  protoplasm. 

Sziics12  has  recently  shown  that  the  toxic  effect  of  copper  sulphate  on 
the  roots  of  Cucurbita  pepo  may  be  inhibited  by  aluminum  chloride  in 
certain  concentrations.  In  this  case  he  used  as  index  of  toxicity  the  ability 
of  the  root  to  react  to  a  geotropic  stimulus  after  it  had  been  removed  from 
the  poisonous  solution.  He  varied  the  presentation  time  of  the  toxic  stimu- 
lus (solution  of  copper  sulphate)  both  alone  and  with  addition  of  aluminum 
chloride,  and  found,  for  the  shorter  time  periods,  that  the  presence  of 
aluminum  inhibited  the  poisonous  action  of  copper.  However,  when  such 

9  Clark,  J.F.,  On  the  toxic  properties  of  some  copper  compounds  with  special  reference  to  Bordeaux 
mixture.     Bot.  Gaz.  33:   26-48.      1902. 

10  Le  Renard,  Alf.,  Influence  du  milieu  sur  la  resistance  du  Pdnicille  crustace  aux  substances  tox- 
iques.     Ann.  Sci.  Nat.  Bot.  IX.   16:  276-336.      1912. 

11  True,  Rodney,  H.,  and  Gies,  W.  J.,  On  the  physiological  action  of  some  of  the  heavy  metals  in 
mixed  solutions.     Bull.  Torr.  Bot.  Club  30:  390-402.      1903. 

12  Sziics,  Joseph,   Experimented   Beitra^e  zu  einer   Theorie  der  antagonistischen  lonenwirkungen. 
Jahrb.  wiss.  Bot.  52:  85-143.      1912. 


TOXICITY  OF  HEAVY  METALS  61 

a  combination  of  the  two  salts  was  allowed  to  act  i&c  longer  periods,  rt 
also  was  toxic  in  many  cases,  and  the  roots  lost  their  ability  to  respond 
afterwards  to  the  geotropic  stimulus.  He  used  concentrations  of  copper 
sulphate  varying  from  o.ooi875n  to  o.o75n,  combined  with  aluminum  chlor- 
ide in  concentrations  varying  from  o.(X>5n  to  o.45n.  The  presentation  time 
of  the  toxic  stimulus  ranged  from  33  minutes  to  26  hours  and  50  minutes^ 
This  writer  also  studied  the  effect  upon  Spirogyra  of  quinine  hydrochloride, 
methyl  violet  and  piperidine  in  combination  with  various  other  substances. 
The  toxicity  of  quinine  hydrochloride  was  altered  by  various  concentrations 
of  other  substances,  being  almost  inhibited  by  aluminum  nitrate,  markedly 
decreased  by  calcium  nitrate,  and  only  slightly  lessened  by  potassium  nitrate. 
Thus,  the  effectiveness  of  these  salts  in  reducing  the  toxicity  of  quinine 
hydrochloride  diminished  with  the  valency  of  the  cation.  Similar  results 
were  obtained  with  the  same  series  of  salts  in  combination  with  methyl 
violet.  The  effect  of  combinations  of  various  substances  with  piperidine 
was,  in  most  cases,  markedly  to  increase  its  toxicity.  Sziics  apparently 
considers  the  antagonistic  action,  as  investigated  by  him,  to  be  due  to  the 
lowering  of  the  rate  of  absorption  of  the  toxic  ion  by  the  presence  in  the 
solution  of  another  ion  of  similar  sign.  He  concludes,  in  summarizing: 
"  Die  Ursache  der  '  antagonistischen  lonenwirkungen  '  liegt  in  alien  von  mir 
untersuchten  Fallen  in  der  gegenseitigen  Beeinflussung  der  Aufnahmege- 
schwindigkeit  zweier  im  gleichen  Sinne  geladener  lonen." 

From  the  results  obtained  in  the  investigations  just  considered,  it  is 
apparent  that  the  toxic  effect  of  the  heavy  metals  on  an  organism  can 
be  modified,  in  some  cases  at  least,  by  the  addition  of  certain  salts  in 
proper  concentration.  True  and  Gies,  and  Sziics,  working  with  higher 
plants,  attribute  this  influence  of  one  salt  on  the  effect  of  another  to  a 
simultaneous  action  of  the  two  salts  upon  the  organism  itself,  while  Clark, 
working  with  fungi,  relates  the  inhibition  of  the  toxic  effect  of  heavy 
metals  in  combination,  to  some  modification  of  the  salts  in  the  solution. 
The  proportions  of  salts  used  in  the  investigations  just  mentioned  were 
widely  different,  and  it  is  of  course  possible  that  the  different  conclusions 
reached  may  have  been  due  to  this  feature.  Furthermore,  as  fungi  and 
higher  plants  so  frequently  react  differently  to  the  same  stimulus,  it  is 
possible  that  one  of  these  two  explanations  might  hold  for  one  group  of 
organisms  and  the  other  for  the  other  group.  The  present  study,  in  which 
a  fungus  was  employed,  was  undertaken  partly  to  throw  light  on  the 
question  just  suggested. 

It  was  the  purpose  of  this  research  to  examine  the  effects  of  the  nitrates 
of  copper,  lead,  zinc,  nickel,  and  aluminum,  upon  the  germination  of 
fungus  spores,  the  salts  of  these  heavy  metals  being  used  both  alone  and 
in  combination  with  the  nitrates  of  calcium  and  magnesium,  to  see  whether 


62  LON  A.  HAWKINS 

the  presence  of  the  lighter  metals  in  various  concentrations  might  or  might 
not  decrease  the  toxic  effect  of  the  heavy  ones.  It  was  also  considered 
worth  while,  when  such  a  decrease  was  found  to  occur,  to  determine  as 
far  as  possible  whether  this  influence  might  be  related  to  a  direct  effect  of 
salts  on  each  other  in  the  solution  or  to  some  modification  of  the  organism 
itself.  Furthermore  the  results  obtained  in  these  experiments  should  throw 
some  light  on  the  problem  of  the  comparative  toxicity  of  the  various  sub- 
stances here  employed,  when  used  alone,  and  thus  upon  the  general  physio- 
logical problem  of  toxicity. 

The  investigation  was  carried  out  at  the  Laboratory  of  Plant  Physiology 
of  the  Johns  Hopkins  University,  and  the  writer's  sincere  thanks  are  due 
to  Professor  Burton  E.  Livingston  for  his  many  helpful  suggestions  and 
valued  assistance  throughout  the  progress  of  the  work. 

ORGANISM. 

The  fungus  spores  used  in  this  research  were  of  the  Gloeosporium  or 
conidial  stage  of  Glomerella  cingulata  (Stonem.)  S.  and  v.  S.,  the  fungus 
causing  the  disease  of  the  apple  known  as  "  bitter  rot."  Not  only  is  the 
fungus  parasitic  upon  the  apple,  but  according  to  Shear  and  Wood,13  it 
is  also  the  cause  of  disease  on  other  plants.  On  the  apple  fruit  it  produces 
brown,  sunken  areas,  usually  nearly  circular  in  shape,  which  may  be  covered 
with  the  fruiting  bodies  of  the  fungus,  the  conidia  being  borne  in  acervuli. 
In  mass,  the  spores  appear  orange-colored  but  have  a  hyaline  appearance 
under  the  microscope.  They  are  usually  ovate  or  oblong  in  shape,  the  two 
diameters  being  12-16  p  and  4-6  p.  Shear  and  Wood  have  shown  that  cer- 
tain strains  of  this  fungus,  when  grown  on  proper  artificial  media,  may 
produce  conidia  for  generation  after  generation,  without  the  interpolation 
of  the  ascogenous  form  at  any  time.  Cultures  of  such  a  strain  were  secured 
from  Dr.  Shear  for  these  experiments,  and  conidia  only  were  produced 
throughout  the  investigation,  which  lasted;  about  eighteen  months,  though 
forty  or  more  generations  must  have  passed. 

Corn  meal  agar  was  used  as  a  medium  for  the  stock  cultures.  This 
was  prepared  by  adding  4  teaspoonsful  of  white  corn  meal  to  one  litre  of 
distilled  water,  which  was  then  allowed  to  stand  at  about  58°  C.  for  one 
hour.  After  filtration,  12.5  g.  of  agar  flour  was  added  to  the  infusion, 
and  the  mixture  was  steamed  for  one  and  one-half  hours.  It  was  then 
re-filtered  and  was  ready  to  tube  and  sterilize.  The  tubes  were  slanted 
and  the  stock  cultures  were  inoculated  in  streaks.  On  this  medium  the 
fungus  produces  but  little  mycelium,  at  or  beneath  the  surface,  and  the 
conidia  are  borne  in  relatively  large,  orange-colored  masses  on  the  surface 

13  Shear,  C.  L.,  and  Wood,  Anna  K.,  Studies  of  fungus  parasites  belonging  to  the  genus  Glomerella. 
U.S.  Dept.  Agric.  Bur.  Plant  Ind.  Bulletin  252.     1913. 


TOXICITY  OF  HEAVY  METALS  63 

of  the  medium  along  the  streak.  The  acervuli  are  visiWe  in  from  two  to 
five  days  after  inoculation. 

Before  the  spores  were  used  in  the  experiments,  the  stock  cultures  were 
allowed  to  grow  undisturbed  from  ten  to  fifteen  days,  a  procedure  which 
insured  a  sufficient  quantity  of  spores  fro.n  a  single  tube  for  the  inocula- 
tion of  an  entire  series  of  experimental  cultures.  Preliminary  tests  indi- 
cated that  spores  from  acervuli  in  the  same  tube,  but  of  different  ages', 
were  not  at  all  uniform  in  viability.  Direct  inoculation  of  the  culture 
dishes  from  the  spore  masses  of  the  stock  tubes  was  thus  shown  to  be 
unsatisfactory,  since  it  was  not  only  desirable  that  all  cultures  contain 
approximately  the  same  number  of  spores,  but  also  that  the  percentage  of 
viability  of  the  spores  in  all  cultures  should  be  as  nearly  alike  as  possible. 
It  also  seemed  desirable  to  avoid  small  pieces  of  agar  and  bits  of  mycelium 
in  the  liquid  cultures,  for  such  contamination  might  influence  the  effect 
of  the  salts  in  the  solutions  on  the  germination  of  the  spores,  as  by 
absorption  or  the  formation  of  new  chemical  compounds.  The  plan  was 
therefore  evolved  of  allowing  the  stock  cultures  to  grow  for  from  ten  to 
fifteen  days,  after  which  period  the  spore  masses  were  carefully  removed, 
with  a  platinum  needle,  to  a  clean  area  on  the  agar  surface,  from  which, 
after  thorough  mixing  in  a  little  heap,  the  inoculations  to  the  water  cul- 
tures were  made.  This  method  usually  resulted  in  satisfactory  uniformity 
in  the  germination  of  the  spores  in  the  various  controls,  from  which 
fact  it  was  concluded  that  all  cultures  thus  made  contained  an  approxi- 
mately equal  number  of  viable  spores,  and  that  any  inhibition  or  modifi- 
cation of  germination  must  be  due-  to  properties  of  the  culture  medium 
rather  than  to  differences  in  the  spores  introduced. 

In  comparing  the  effect  of  the  various  media  upon  the  spores,  the  main 
criterion  was  the  presence  or  absence  of  germination  after  a  period  of 
eighteen  hours.  In  many  cases,  however,  germination  was  more  or  less 
modified,  as  in  the  production  of  swollen  tubes  and  other  abnormalities, 
and  such  modifications  of  germinal  activity  needed  frequently  to  be  taken 
into  account.  As  has  been  indicated,  it  was  seldom  necessary  to  consider 
the  percentage  of  normal  germination  which  occurred,  but  in  many  cases 
the  proportion  of  abnormal  to  normal  growth  was  approximately 
determined. 

These  Gloeosporiurh  spores  possess  several  very  favorable  features  for 
such  an  investigation  as  the  present.  They  are  readily  wetted  by  water 
and  aqueous  solutions  and,  being  slightly  heavier  than  water,  they  sink 
quickly  to  the  bottom  of  a  hanging  drop.  They  germinate  readily  in  dis- 
tilled water  in  from  three  to  four  hours  and  produce  long  filaments  in 
eighteen  hours,  a  feature  which  is  of  considerable  advantage  here,  for  it 
is  quite  conceivable  that  the  influence  of  various  chemical  compounds  on 


64  LON  A.  HAWKINS 

each  other  and  on  the  germinating  spores  might  be  considerably  altered 
if  nutrient  salts  were  present  in  the  solution.  In  view  of  these  considera- 
tions these  experiments  were  carried  out  without  the  use  of  nutrient  media. 

METHODS. 

The  salts  used  in  these  experiments  were  "  Baker's  analysed  "  chemicals, 
procured  in  the  original  packages.  Stock  solutions  of  the  different  salts, 
from  which  the  experimental  solutions  were  afterwards  prepared,  were 
made  up  in  o.2m,14  o.5m,  or  molecular  concentration.  In  preparing  the 
stock  solutions  the  salts  were  weighed  in  glass-stoppered  weighing  bottles 
directly  from  freshly  opened  packages  and  were  dissolved  in  volumetric 
flasks.  These  solutions  were  then  made  up  to  the  required  volume  at  a 
temperature  of  15°  C.  They  were  stored  in  Jena  glass  bottles  which  had 
been  carefully  washed  with  saturated  solution  of  potassium  dichromate  in 
sulphuric  acid,  steamed  for  half  a  day,  again  washed  with  distilled  water 
and  finally  allowed  to  soak  in  distilled  water  for  a  month  or  more,  to 
remove  any  soluble  matter  which  might  be  present.  Distilled  water  from 
a  still  with  tin  lined  boiler  and  condenser  was  used  in  making  the  stock 
solutions  as  well  as  in  diluting  them  for  the  cultural  work.  Preliminary 
tests  showed  that  the  spores  germinated  as  well  in  water  from  this  still 
as  in  more  nearly  pure  water  distilled  from  potassium  permanganate  solu- 
tion, using  a  hard  glass  flask  and  a  block  tin  condenser. 

The  stock  salt  solutions  were  diluted  to  the  concentrations  required  in 
making  up  the  solutions  for  the  experiments,  by  pipetting  out  the  proper 
amount  into  a  hard  glass  beaker  and  then  adding  the  necessary  distilled 
water  from  a  burette.  The  concentrations  of  these  solutions  were  so  cal- 
culated that  the  culture  solutions  could  be  prepared  without  the  measure- 
ment of  less  than  0.5  cc.  in  any  case.  Thus,  errors  that  might  have  arisen 
in  attempting  to  read  hundredths  of  a  cubic  centimeter  on  a  burette  gradu- 
ated only  to  tenths,  were  obviated. 

In  making  up  a  series  of  cultures,  the  two  salt  solutions  which  were  to 
be  combined  were  separately  diluted  to  twice  the  concentration  finally 
desired,  and  were  then  placed  in  burettes.  From  these  were  prepared,  with 
addition  of  water  as  needed,  the  combinations  and  concentrations  actually 
used  in  the  experiments.  These  mixtures,  in  volumes  of  10  cc.  or  more, 
were  made  in  small  flasks  (of  about  75  cc.  capacity),  a  flask  being  pro- 
vided for  each  of  the  different  combinations  as  well  as  one  for  the  control. 
The  latter  solution  usually  contained  the  salt  of  the  heavy  metal  alone. 

From  each  of  the  flasks  just  mentioned  a  small  portion  of  solution  (about 

14  The  letter  nt  is  used  throughout  this  paper  to  denote  molecular,  a  concentration  of  a  single  gram 
molecule  in  a  liter  of  solution.  There  seems  to  be  considerable  confusion  in  this  important  matter  of 
defining  solutions,  some  chemists  employing  the  letter  n  to  denote  molecular,  although  the  latter  has 
long  been  used  to  denote  chemically  equivalent. 


TOXICITY  OF  HEAVY  METALS  65 

a  cubic  centimeter)  was  placed  in  a  separate  glass  cylinder  (2  cm.  high 
and  3  cm.  in  diameter),  to  which  spores  from  a  stock  culture  were  then 
transferred.  These  inoculations  were  made  in  order,  beginning  with  the 
weakest  solution  of  the  lighter  metal.  A  platinum  needle  was  used  for 
this  purpose,  flamed  and  washed  to  sterilize  and  clean  it  after  each 
inoculation. 

In  each  case  the  tip  of  the  needle  was  dipped  a  single  time  in  the  well 
mixed  mass  of  spores  which  had  been  prepared  on  the  agar  surface,  as 
already  described,  and  the  spores  that  adhered  were  washed  off  in  the  culture 
solution.  Thus,  approximately  the  same  number  of  spores  were  inoculated 
into  all  of  the  glass  dishes  and  the  solutions  were  then  ready  for  the 
preparation  of  the  hanging  drops. 

The  drop  cultures  were  made  after  much  the  same  method  as  that 
described  by  Clark.15  Van  Tieghem  cells  were  used,  small  cylinders  of 
glass  tubing,  with  ground  ends,  9  mm.  high  and  12  mm.  in  diameter,  which 
were  cemented  to  ordinary  microscope  slides  by  means  of  beeswax.  Two 
cells  were  affixed  to  each  slide.  The  culture  solutions  in  the  glass  dishes, 
into  which  spores  had  already  been  inoculated,  were  thoroughly  mixed 
with  a  glass  rod.  By  means  of  this  rod,  a  drop  of  the  liquid  was  then 
placed  upon  a  flamed  cover  glass. 

A  small  drop  of  the  culture  solution  from  the  corresponding  flask  with- 
out spores  was  placed  in  the  bottom  of  the  Van  Tieghem  cell  and  the  cover 
bearing  the  drop  culture  was  inverted  over  it.  Duplicate  drop  cultures 
were  made  from  each  concentration  of  solution,  both  cultures  being  placed 
on  the  same  slide.  As  has  been  shown  by  Clark,  the  presence  at  the 
bottom  of  the  culture  cell  of  a  small  amount  of  the  same  solution  as  that 
from  which  the  hanging  drop  is  composed  prevents  evaporation  from  the 
drop  and  hence  obviates  marked  alteration  in  its  concentration,  even  if  the 
cultures  remain  in  the  thermostat  for  a  considerable  time.  Without  this 
precaution  the  solution  contained  in  the  hanging  drop  is  apt  to  become 
markedly  more  concentrated  during  the  period  of  an  experiment,  which 
might  lead  to  erratic  results.  The  covers  were  sealed  in  position  with 
petrolatum.  It  was  not  found  necessary  to  take  the  precaution  recom- 
mended by  Clark,  of  first  allowing  the  expanding  air  to  escape  through 
a  small  opening  in  the  seal,  possibly  because  the  temperature  of  the  thermo- 
stat here  used  was  only  a  little  above  the  temperature  at  which  the  prepara- 
tions were  made. 

For  ease  in  handling  the  cultures,  the  slides  were  placed  in  sheet  metal 
trays,  which  could  be  piled  one  upon  another  in  the  thermostat  so  as  to 
form  a  rack.  These  trays  were  15  cm.  wide  and  20  cm.  long,  with  vertical 

15  Clark,  J.  P.,  On  the  toxic  effect  of  deleterious  agents  on  the  germination  and  development  of  cer- 
tain filamentous  funtn.     Bot.  Gaz.  28:  289-327,  378-404.     1890. 


66  LON  A.  HAWKINS 

flanges  at  the  ends,  the  flanges  extending  upward  about  1.3  cm.  and  down- 
ward about  0.4  cm.  They  were  so  bent  that  the  lower  flanges  of  one 
tray  fitted  outside  the  upper  flanges  of  the  next  lower  one,  and  many 
trays  could  thus  be  arranged  in  a  compact  and  rigid  pile  without  any 
disturbance  to  the  slides.  The  bottom  of  each  tray  was  perforated  with 
circular  openings  about  1.5  cm.  in  diameter  and  0.5  cm.  apart,  to  facilitate 
circulation  of  air.  Each  tray  carried  fourteen  slides.  The  slides  were 
always  transferred  to  and  from  the  thermostat  by  means  of  the  trays,  a 
whole  series  of  cultures  being  thus  moved  together. 

The  cultures  were  kept  during  germination  in  an  electrically  heated  and 
automatically  regulated  thermostat,  in  which  the  temperature  was  main- 
tained at  or  near  25°  C.  As  the  temperature  of  the  room  in  which  the 
thermostat  was  placed  sometimes  rose  above  25°,  it  was  necessary  to 
install  apparatus  for  absorbing  heat  at  such  times.  To  accomplish  this, 
several  coils  of  thin-walled  copper  tubing  carrying  a  continuous  stream  of 
tap  water  were  placed  at  the  top  of  the  thermostat,  surrounding  a  small 
motor-driven  fan,  the  latter  insuring  air  circulation.  The  air  of  the 
chamber  then  tended  to  assume  a  temperature  several  degrees  below  that 
of  the  laboratory,  and  the  thermostat  acted  as  though  standing  in  a 
cold  room. 

EXPERIMENTATION, 

In  these  studies,  any  renewed  activity  in  the  protoplasm  of  the  spore 
was  considered  as  germination.  Several  forms  of  such  renewed  activity 
are  exhibited  by  the  conidia  here  employed.  Without  any  alteration  in 
size  or  shape,  a  portion  of  the  spore  may  become  nearly  or  quite  opaque, 
thus  appearing  dark  brown  or  black  by  transmitted  light.  A  papilla  may 
•be  formed  at  any  point  on  the  surface.  Such  papillae  may  or  may  not 
enlarge  to  form  rounded  bodies,  and  may  either  remain  hyaline  or  become 
apparently  darkened.  Papillae  may  enlarge  to  form  irregularly  shaped 
bodies  or  <may  extend  outward  as  markedly  thickened  tubes.  Lastly,  the 
growing  papillae  may  take  the  form  of  slender  tubes.  The  latter  type  of 
germination  always  occurs  in  distilled  water  and  the  tubes  attain,  in  a 
period  of  eighteen  hours,  a  length  at  least  twice  as  great  as  that  of  the 
spore  itself.  In  the  following  treatment  germination  will  be  considered 
"  normal  "  wherever  tubes  of  the  last  mentioned  type,  at  least  twice  the 
length  of  the  spore,  were  produced  in  eighteen  hours. 

In  the  present  section  will  be  considered  the  various  effects  upon  germ- 
ination brought  about  in  the  presence  of  the  following  salts  either  alone 
or  in  certain  combinations : 

Ca(NO3)2  Pb(NO3)2  Ni(NO,,)2 

Mg(NO3)2  Zn(NO3)2  Cu(NO3)2 

KNO3  A1(NO3)3  CuSO4' 


TOXICITY  OF  HEAVY  METALS  67 


TOXICITY    OF    SALT    COMBINATIONS 

Calcium,  Magnesium  and  Potassium. 

It  requires  a  relatively  high  concentration  of  calcium,  magnesium  or 
potassium  nitrate  to  inhibit  the  germination  of  these  spores.  With 
Ca(NO3)2  normal  germination  was  found  in  the  0.5111  solution  and 
swollen  tubes  were  abundant  in  o.6m.  At  a  concentration  of  o./m,  how- 
ever, none  of  the  spores  germinated  in  any  of  the  four  series  of  duplicate 
cultures  which  were  carried  out.  The  concentration  of  this  salt  which 
just  prevents  germination  appears,  therefore,  to  lie  between  o.6m  and 
o./m.  Magnesium  nitrate  is  similar  to  that  of  calcium  in  its  effect.  With 
this  salt  the  spores  germinated  normally  at  a  concentration  of  0.4111,  and 
local  swellings  of  the  spore  wall  and  swollen  tubes  were  produced  in  0.5111 
solution,  while  no  germination  was  evident  when  a  o.6m  concentration  was 
employed.  The  concentration  at  which  Mg(NO3)2  just  inhibited  germin- 
ation thus  seems  to  lie  between  o.5m  and  o.6m.  In  solutions  of  KNO3 
the  spores  germinated  normally  with  a  concentration  of  0.9111  and  formed 
local  swellings  of  the  spore  wall  with  molecular  concentration.  As  the 
latter  was  the  highest  concentration  here  used,  the 
point  at  which  KXCX  inhibits  germination  was  not 
determined. 

The  exceedingly  high  .concentrations  of  these  salts, 
which  were  found  necessary  to  inhibit  germination, 
made  it  seem  possible  that  they  might  be  without  toxic 
effect  upon  the  spores  ;  the  inhibition  of  germination 

which  was  observed  in  high  concentrations  might  have 

.-«..-,  .  in?  spores  from  i.6m 

been  the  result  of  the  high  osmotic  pressure  exerted      sucrose   solution,    x 

by  the  medium.     To  obtain  evidence  on  this  point  the      550. 
germination  of  the   spores  was  tested  in  cane  sugar 

solution  at  various  concentrations.  A  o.2m  stock  solution  was  prepared 
from  granulated  sugar  which  had  been  previously  pulverized  and  desic- 
cated ;  the  different  concentrations  used  in  the  cultures  were  prepared  from 
this.  The  spores  germinated  normally  in  concentrations  of  cane  sugar 
up  to  and  including  i-4m,  considerable  germination  in  the  form  of  local 
swellings  (shown  in  fig.  i)  was  found  in  the  i.6m  solution,  and  no  germina- 
tion occurred  with  i.8m  concentration.  The  concentration  of  cane  sugar 
which  just  inhibits  germination  lies  then  between  i.6m  and  i.8m. 

It  will  be  observed  that  a  i.6m  solution  of  sucrose  has  about  the  same 
effect  upon  the  germination  of  these  Gloeosporium  spores  as  do  o.6m,  0.5111 
and  molecular  solutions  of  calcium,  magnesium  and  potassium  nitrates 


68  LON  A.  HAWKINS 

respectively.  Calculations16  of  the  osmotic  pressures  of  these  four  solu- 
tions give  51.36,  29.13,  26.00  and  39.31  atmospheres,  respectively,  from 
which  numbers  it  becomes  obvious  that  the  osmotic  concentration  of  the 
sugar  solution  was  much  greater  than  that  of  the  other  three.  It  is  there- 
fore suggested  that,  unless  the  wall  and  protoplasm  of  the  spores  here 
employed  be  readily  permeable  to  cane  sugar  (which  seems  highly  improb- 
able), the  inhibiting  effects  observed  with  the  salt  solutions  are  not  to  be 
related  primarily  to  their  osmotic  properties. 

The  relatively  high  osmotic  pressure  existing  within  the  spores  here 
dealt  with,  as  indicated  by  the  data  just  given,  renders  it  unnecessary  to 
consider  osmotic  pressure  as  a  factor  in  bringing  about  the  modifications 
and  inhibitions  of  germination  which  are  next  to  be  considered ;  the  con- 
centrations of  the  various  salt  solutions  employed  were  always  far  too 
low  to  produce  any  removal  of  water  from  the  cells.  Similarly,  the 
toxicity  of  calcium,  magnesium  and  potassium  nitrates  is  so  very  slight 
as  not  to  require  any  consideration  in  connection  with  the  combinations  of 
these  salts  with  those  of  the  heavy  metals ;  in  these  combinations  the 
salts  of  the  lighter  metals  have  not  here  been  used  in  concentrations  higher 
than  o.im. 

The  influence  of  the  nitrates  of  calcium,  magnesium  and  potassium  upon 
the  toxicity  of  salts  of  the  heavy  metals  will  now  receive  attention. 


Copper. 

As  was  to  be  expected,  the  effect  of  Cu(NO3)2  upon  the  germination  of 
these  spores  was  found  to  be  widely  different  from  that  of  Ca(NO3)2.  No 
germination  was  ever  found  in  concentrations  of  the  copper  salt  alone 
higher  than  0.00006111,  and  it  was  only  in  an  occasional  culture  that  one 
or  two  spores  were  observed  producing  local  swellings  with  that  concen- 
tration. Local  swellings  of  the  spore  walls  and  swollen  tubes  were  fre- 
quently found  with  0.00004111  and  with  o.oooo26m  concentration,  the  next 
lower  one  which  was  here  used;  with  0.00002111  solution  of  Cu(NO3)2 
germination  was  about  as  in  distilled  water. 

Copper  nitrate  was  combined  with  Ca(NO3)2,  at  several  concentrations 
of  the  copper  salt  well  above  that  at  which  the  latter  was  non-toxic  when 
used  alone.  In  a  series  of  combinations  of  o.oooim  Cu(NO3)2  with  dif- 

18  Morse,  H.  N.,  and  Holland,  W.  W.,  The  osmotic  pressure  of  cane  sugar  solutions  at  25°.  Amer. 
Chem.  Jour.  41:  I-IQ.  IQOQ.  The  osmotic  pressure  of  molecular  cane  sutfar  was  measured  directly  by 
these  writers  and  the  osmotic  pressure  of  1.6  m.  cane  sugar  as  given  above  has  been  calculated  from  their 
tables.  The  result  is  probably  low,  for  they  found  that  the  ratio  of  observed  to  calculated  osmotic  pres- 
sure increases  with  the  concentration.  For  the  gas  pressure  formula,  by  which  the  osmotic  pressure 
of  the  two  electrolytes  is  calculated,  see  Landolt,  H.,  Bornstein,  R.,  and  Meyerhoffer,  W.,  Physikalische- 
chemische  Tabellen,  3te  Aufllaije.  Berlin.  1005;  page  24. 


TOXICITY  OF  HEAVY  METALS  69 

ferent  concentrations  of  the  calcium  salt,  ranging  from  0.00003 125m  to 
o.ooo625m,  the  spores  germinated  readily  in  those  cultures  where  the  con- 
centration of  Ca(NO3)2  was  0.0000625111  or  higher.  With  the  last  named 
concentration  many  local  swellings  were  found,  and  also  short  swollen 
tubes  (see  fig.  2),  much  as  in  the  0.00004111  concentration  of  Cu(NO3)2 
alone.  With  the  next  lower  concentration  of  the  calcium  salt,  0.00003 125m, 
no  germination  occurred. 

From  these  facts  it  appears  that  the  toxicity  of  o.oooim  Cu(NO3)2  solu- 
tion is  so  reduced  as  to  be  physiologically  equivalent  to  a  0.00004111  solution 
of  the  same  salt,  by  the  addition  to  the  former  solution  of  five  molecules 
of  Ca(NO3)2  for  every  eight  molecules  of  Cu(NO3)2  present.  The  addi- 
tion of  the  lighter  metal  in  this  proportion  pro- 
duces the  same  effect  as  though  the  o.oooim  cop- 
per solution  had  been  diluted  to  two  and  a  half 
times  its  original  volume. 

Copper  sulphate  exhibits  about  the  same  tox- 
icity toward  these  Gloeosporium  spores  as  does 
the  nitrate.  A  series  of  combinations  of  this  salt 
with  Ca(NO3)2,  quite  similar  to  the  series  with  Fig.  2.  Germinating 
Cu(NO3)2  just  described,  gave  no  germination  spores  from  combination 
in  solutions  containing  0.00003125111  concen-  solution  containing 

trations    of    the    calcium    salt,    while    germina-      °-000°6m     Ca(NO3)2     and 
.     .  '  o.oooim  Cu(N03)2,    X65o. 

tion  occurred  in  some  of  the  cultures  con- 
taining 0.0000625111  Ca(NO3)2.  In  general,  the  effect  of  CuSO4,  either 
alone  or  in  combination  with  Ca(NO3)2,  was  practically  the  same  as  was 
that  of  Cu(NO3)2.  This  furnishes  some  additional  evidence  toward  the 
already  rather  firmly  established  conclusion,  that  the  toxicity  of  copper 
salts  is  due  to  the  cations,  and  it  also  indicates  the  probability  that  con- 
siderations bearing  upon  this  toxicity  need  deal  only  with  the  cations. 

A  series  of  experiments  was  carried  out  using  o.ooo/mi  Cu(NO3)2  in 
combination  with  concentrations  of  Ca(NO3)2  ranging  from  0.00025111  to 
o.O25m.  The  spores  in  these  combinations  germinated  readily  in  the  pres- 
ence of  Ca(NO3)2  in  concentrations  ranging  from  o.ooim  to  0.025111,  but 
no  germination  was  found  in  combinations  with  0.0005111  or  0.00025111  of 
the  calcium  salt.  The  form  and  abundance  of  germination  with  o.ooim 
Ca(NO3)2  was  here  much  the  same  as  that  found  in  0.00004111  of  the  copper 
salt  alone.  Here  a  ratio  of  five  molecules  of  the  calcium  salt  to  two  of 
the  copper  salt,  the  latter  occurring  in  the  solution  at  a  concentration  of 
0.0004111,  reduces  the  toxicity  of  the  Cu(NO3)2  so  as  to  produce  an  effect 
on  spore  germination  equivalent  to  that  exercised  by  a  0.00004111  solution 
of  the  copper  salt  alone.  In  other  words,  addition  to  a  0.0004111  Cu(NO3)2 
solution,  of  Ca(NO8)a  in  the  molecular  ratio  of  five  of  the  latter  to  two  of 


70  LON  A.  HAWKINS 

the  copper  Salt,  has  the  same  effect  as  diluting  the  Cu(NO3)2  solution  to 
ten  times  its  original  volume. 

A  third  series  of  combinations  of  the  same  two  salts,  but  with  higher 
concentrations,  was  carried  out  in  a  manner  somewhat  different  from  that 
employed  in  the  preceding  series.  Here  the  concentration  of  Ca(NOs)8 
was  the  same  (o.o5m)  in  all  of  the  cultures,  while  that  of  the  copper  salt 
varied.  The  concentrations  of  Cu(NO3)2  ranged  from  0.00025111  to  o.oim, 
and  the  spores  germinated  in  all  the  concentrations  used  except  the  highest. 
With  a  concentration  of  o.ooSm  Cu(NO3)2  germination  was  similar  to 
that  found  with  0.00004111  of  the  copper  salt  without  the  addition  of 
Ca(NO3)2.  From  this  series  it  appears  that  addition  to  the  Cu(NO3)2  solu- 
tion here  used  (o.ooSm),  of  about  six  molecules  of  Ca(NO3)2  for  each  mole- 
cule of  the  copper  salt  present  in  the  solution,  reduces  the  toxicity  of  the 
latter  compound  in  the  same  way  as  though  the  solution  had  been  diluted 
to  two  hundred  times  its  original  volume.  It  appears  as  though  the  presence 
of  Ca(NO3)2,  in  this  molecular  proportion  of  about  6  to  i,  altered  the 
relations  between  spores  and  solution  so  that  only  a  two-hundredth  part 
of  the  copper  nitrate  actually  present  was  effective  to  modify  or  retard 
the  germination  processes. 

A  series  of  combinations  of  Cu(NO3)2  with  KNO3  was  carried  out, 
in  which  'a  constant  concentration  of  the  potassium  salt,  0.05111,  was  used 
in  combination  with  Cu(NO3)2  in  concentrations  ranging  from  0.0002111  to 
o.oim.  The  spores  germinated  in  concentrations  up  to  and  including  0.002111 
of  the  copper  salt.  In  the  last  mentioned  concentration,  the  form  of 
germination  was  quite  similar  to  that  found  in  a  0.00004111  solution  of  the 
copper  salt  alone.  Thus  in  a  solution  containing  KXO3  at  a  0.05111  con- 
centration and  Cu(NO3),  at  a  0.002111  concentration  (a  molecular  ratio 
of  25  of  the  potassium  salt  to  i  of  the  other),  the  toxicity  of  the  copper 
salt  is  decreased  to  a  magnitude  only  one  fiftieth  as  great  as  is  that  shown 
by  this  concentration  of  the  copper  salt  alone. 

The  results  of  the  three  series  of  combinations  of  copper  nitrate  with 
calcium  nitrate  which  have  been  described,  are  summarized  in  table  I, 
together  with  certain  other  data  which  require  consideration. 

From  the  data  just  given  the  question  arises,  whether  the  influence  of 
calcium  nitrate  in  reducing  the  toxicity  of  the  copper  salt  may  be  due  to 
a  direct  effect  produced  in  the  solution  or  to  some  change  brought  about 
in  the  spore  itself.  Kronig  and  Paul  [97],  in  considering  an  effect 
similar  to  this,  in  which  the  toxicity  of  HgCl2  upon  Bacillus  anthracis 
was  decreased  by  the  addition  of  NaCl,  concluded  that  the  change  thus 
brought  about  is  due  to  depression  of  the  ionization  of  the  salt  of  the 
heavy  metal.  Such  a  supposition  cannot  apply  in  the  present  case,  how- 
ever; that  alteration  in  the  ionization  of  Cu(NO3)2  cannot  be  the  cause 


TOXICITY  OF  HEAVY  METALS  71 

of  the  diminution  of  its  toxicity  as  here  observed,  is  clearly  shown  by 
simple  calculation  according  to  the  isohydric  principle.  The  percentage  of 
dissociation  of  Cu(NO3)2  in  the  mixtures  here  employed  should  be  deter- 
mined by  the  concentration  of  the  NO3  radical.  Following  this  generaliza- 
tion, the  data  given  in  table  I  (C,  a)  have  been  obtained.  Seventy-six  per 
cent,  of  the  copper  salt  of  the  first  combination  is  seen  to  be  dissociated. 


TABLE  I 


•  . 

Series 
I 

Series 
II 

Series 
III 

A.  Cu(XO3)2  present  in  the 
medium,  molecular.  .  .  t 

a.  Common  fraction  

TT5 

J3U<T 

i<To<nr 

b  Decimal  fraction  

o  .008 

o  .0004 

0  .0001 

B.  Ca(NO3)2  required  to  al- 
low    same     kind     and 
amount  of  germination 
as  occurs  in  0.00004111 
(m/25ooo)        Cu(X03)2 

without  PafVO  1 

a.  Common  fraction,  mole- 
cular   

* 

TIFffTf 

T^ffff 

b.  Decimal  fraction,  mole- 
cular   

0.05 

O  .OOI 

o  .0001 

c.  Number  of  molecules  of 
Ca(XO3)2  for  each 
molecule  of  Cu(XO3)2. 

6.25 

2  .50 

0.63 

C.  Concentration    of    disso- 
ciated  Cu(XO3)2   pres- 
ent  in   mixture,    mole- 
cular   

a.  According  to  the  isohy- 
dric principle  

o  .0061 

o  .0004 

o  .0001 

b.  From     potentiometer 
measurements 

o  .0058 

o  .0004 

o  .0001 

D.  Concentration  of  disso- 
ciated  Cu(XO3)2   in 
physiologically  equiva- 
lent   solution    of    this 
salt  alone,  molecular  .  . 

From     conductivity  meas- 
urements (Jones' 
tables)  

o  .00004 

o  .00004 

o  .00004 

Now,  if  only  one  per  cent,  of  the  copper  here  actually  present  in  the 
mixture  were  ionized,  the  concentration  of  the  Cu  ion  would  correspond 
to  that  in  an  o.ooooSm  solution  of  Cu(NO3),  alone,  which,  as  has  been 
shown,  is  stronger  than  is  necessary  to  inhibit  spore  germination.  It  is 
therefore  apparent  that  the  decrease  in  the  toxicity  of  the  copper  salt  shown 
in  these  combinations  with  Ca(NO3)2  is  not  to  be  related  to  decreased 
ionization  of  Cu(NO3)2. 

Another  theory  to  account  for  a  similarly  decreased  toxicity  of  HgCU 


72  LON  A.  HAWKINS 

in  the  presence  of  NaCl  was  advanced  by  Clark  [01],  who  considered  that 
a  double  salt  was  formed,  such  as  Na2HgQ4,  and  supposed  this  to  be 
only  slightly  dissociated  in  the  presence  of  an  excess  of  NaCl.  This  writer 
also  worked  with  various  copper  salts  in  the  presence  of  KNO3,  K2SO4, 
NH2SO4,  etc.,  and  suggested  that  the  decreased  toxicity  of  the  copper  salts, 
which  was  uniformly  observed  in  such  combinations,  was  likewise  due  to 
formation  of  double  salts. 

In  view  of  the  work  of  Hosford  and  Jones,17  on  the  dissociation  of 
double  salts  in  dilute  solution,  it  seems  very  improbable  that  the  lowering 
of  the  toxicity  of  Cu(NO3)2  brought  about  by  Ca(NO3)2,  as  shown  in 
the  present  study,  is  due  to  decreased  ionization  of  the  toxic  compound, 
brought  about  by  double  salt  formation.  It  is,  however,  possible  to  obtain 
direct  evidence  on  this  question  by  comparing  the  concentrations  of  copper 
ions  in  the  various  solutions,  with  and  without  the  calcium  salt.  The  per- 
centage of  dissociation  occurring  in  simple  solutions  of  Cu(NO3)2  has  been 
determined  experimentally  for  many  concentrations,  and  these  data  are 
available  in  published  tables.18  From  such  data  may  be  derived  the  con- 
centration of  copper  ions  present  in  any  solution  containing  only  Cu(NO3)2, 
but  no  published  data  are  yet  available  from  which  might  be  ascertained 
the  concentrations  of  copper  ions  in  the  binary  mixtures  here  dealt  with. 

The  determinations  here  required  of  the  relative  concentrations  of  copper 
ions  in  solutions  of  Cu(NO3)2  with  and  without  addition  of  the  calcium 
salt,  may  be  made  by  means  of  the  potentiometer.10  With  this  instrument 
the  solutions  involved  in  table  I  were  tested,  and  the  differences  in 
electrical  potential  between  the  combination  solutions  of  Cu(NO3)2  and 
Ca(NO3)2  and  the  corresponding  simple  solutions  of  Cu(NO3)2  were 
measured.  The  two  solutions  to  be  tested  (for  example,  the  simple  solu- 
tion containing  o.ooSm  Cu(NO3)2  and  the  combination  solution  containing 
o.ooSm  Cu(NO3)2  and  o.O5m  Ca(NO3)2)  were  placed,  respectively,  in  the 
two  arms  of  a  U-tube,  separated  below  by  a  saturated  solution  of  ammonium 
nitrate.  Into  each  arm  of  the  U-tube  was  introduced  a  copper  electrode 
and  these  were  connected  with  a  potentiometer  (Leeds  and  Northrup,  type 
K).  The  potentiometer  was  so  calibrated  that  the  difference  in  voltage 
between  the  two  copper  solutions  might  be  read  directly  on  the  instrument. 


17  Hosford,  H.  H.,  and  Jones,  Harry  C.,  Conductivity,  temperature  coefficients  of  conductivity  and 
dissociation  of  certain  electrolytes.     Amer.  Chem.  Jour.  46:   240-278.      ign. 

18  Jones,    Harry   C.,    Electrical   conductivity,   dissociation   and    temperature   coefficients   of   conduc- 
tivity,  from  zero  to  sixty-five  degrees,   of  aqueous  solutions  of  a   number  of  salts  and   organic  acids. 
Carnegie   Institution  of  Washington  Publ.   170.     Washington.      1012. 

19  Loomis,  N.  E.,  and  Acree.  S.  F.,  A  study  of  the  hydrogen  electrode,  of  the  calomel  electrode,  and 
of  contact  potential.     Amer.  Chem.  Jour.  46:  585-620.      191 1. 

It  is  a  pleasure  to  acknowledge  here,  that  the  suggestion  of  this  method,  as  applicable  to  the  prob- 
lem in  hand,  is  due  to  Professor  S.  F.  Acree,  of  the  Chemistry  Department  of  the  Johns  Hopkins  Uni- 
versity, and  that  much  valuable  help  in  making  the  determinations  was  received  from  him  and  from 
Dr.  C.  N.  Myers.  This  part  of  the  work  was  carried  out  at  the  chemical  laboratory. 


TOXICITY  OF  HEAVY  METALS  73 

The  copper  electrodes  were  freshly  plated  and  calibrated,  "for  subsequent 
correction  of  the  readings,  with  reference  to  any  difference  in  potential 
which  might  exist  between  them  when  both  were  placed  in  the  simple 
Cu(XO3)2  solution. 

The  difference  in  concentration  of  copper  inns  in  the  two  solutions  thus 
compared  was  calculated  from  the  observed  difference  in  electrical  potential 
by  the  following  formula : 

V  =  0.059 1    logio-?^ 
*«t 

in  which  V  is  the  observed  Electromotive  force,  in  volts ;  C  and  Q  are 
the  concentrations,  respectively,  of  the  copper  ions  in  the  two  solutions, 
and  the  quantity  0.0591  is  a  constant  (Loomis  and  Acree  [n],  page  591). 
In  each  of  the  tests  with  which  we  are  at  present  concerned  three  entirely 
separate  determinations  were  made,  for  each  of  which  the  reading  was 
several  times  repeated,  and  the  results  were  in  excellent  agreement.  It  was 
found  that  the  difference  in  electric  potential  between  the  0.008111  solution 
of  Cu(XO:j)2  and  the  same  solution  containing  also  a  0.05111  concentration 
of  Ca(XO.j)2,  was  0.006  volt,  the  simple  solution  having  the  higher 
potential.  From  this  observation  it  appears,  by  substitution  in  the  formula 
given  above,  that 

C 
r  =1.26, 

^i 

or  that  the  concentration  of  copper  ions  in  the  combination   solution  is 

— ^,  or  0.794,  as  great  as  that  in  the  simple' one.     Xow,  from  Jones'  [12] 

conductivity  tables  (page  57),  it  is  found  by  interpolation  that  the 
Cu(XO:!)2  in  our  simple  solution  (0.008111)  is  about  90.6  per  cent,  disso- 
ciated at  25°  C.  It  thus  appears  that  the  Cu(XO3)2  in  the  combination 
solution  here  considered  must  be  dissociated  to  an  extent  equal  to  0.794  X 
90.6,  or  71.9  per  cent. 

It  has  been  shown  above  (page  71)  that  the  Ca(NO3)2  in  this  particular 
combination  should  decrease  the  dissociation  of  the  copper  salt,  on  account 
of  the  common  ion,  to  76  per  cent.  There  is  thus  a  difference  of  about 
4  per  cent,  between  the  dissociation  of  the  Cu(XO3)2  in  this  mixture  as 
calculated  from  the  isohydric  principle  and  that  derived  from  the  potenti- 
ometer measurements.  This  difference  may,  of  course,  be  due  to  the  forma- 
tion of  a  double  salt,  following  the  hypothesis  of  Clark  already  mentioned, 
but  it  makes  no  difference  in  the  present  discussion  whether  it  be  76  or 
72  per  cent,  of  the  Cu(XO3)2  that  is  here  dissociated;  as  has  been  stated, 
the  solution  would  still  be  toxic  enough  to  inhibit  germination  in  these 
spores  if  only  one  per  cent,  of  the  total  Cu(XO3)2  were  dissociated. 

The  remaining  two  concentrations  of  Cu(XO3)2  (0.0004111  and  o.oooirn, 

PHYSIOLOGICAL   RESEARCHES  VOL.    I,   NO.   2,   SERIAL  NO.    2, 
AUOUST,    1913. 


74  LON  A.  HAWKINS 

see  table  I),  with  and  without  additional  Ca(NO3)2,  were  also  subjected 
to  potentiometer  determinations  of  the  concentrations  of  copper  ions  present 
therein.  In  neither  case  was  there  any  difference  in  electrical  potential 
between  the  two  corresponding  solutions.  It  is  therefore  clear  that  at  least 
99  per  cent,  of  the  Cu(NO3)2  is  to  be  considered  as  dissociated  in  these 
solutions,  whether  the  calcium  salt  be  present  or  not  (see  table  I,  C,  b). 

From  the  foregoing  considerations,  it  seems  quite  clear  that  the  influence 
of  Ca(NO3)2  in  reducing  the  toxic  effect  of  Cu(NO3)2  on  the  germination 
of  the  spores  here  employed  is  not  at  all  to  be  related  to  any  changes 
brought  about  in  the  solution  itself  by  the  addition  of  the  calcium  salt. 
It  appears  that  this  antitoxic  or  antagonistic  influence  must  be  effective 
upon  the  spores  themselves,  so  altering  them  that  they  become  capable  of 
germination  in  solutions  whose  concentration  of  free  copper  ions  would 
inhibit  this  process  were  it  not  for  the  presence  of  the  calcium  salt. 

Whether  the  copper  enters  the  spores  and  exerts  its  toxic  action  directly, 
through  some  alteration  in  the  protoplasm,  or  whether  this  toxic  influence 
is  exerted  primarily  upon  the  spore  walls  or  upon  the  surface  film  of  the 
protoplasm,  thus  perhaps  creating  some  disturbance  in  physico-chemical 
equilibrium  that  may  subsequently  be  propagated  inward,  is  a  question  for 
the  answering  of  which  no  evidence  is  yet  at  hand.  Nevertheless,  the  present 
studies  have  clearly  demonstrated  that  the  presence  of  Cu(XO3)2  alone 
in  the  medium  inhibits  germination  if  the  concentration  be  above  a  very 
low  limit,  produces  markedly  altered  forms  of  renewed  activity  if  the  con- 
centration is  somewhat  lower.,  and  allows  normal  germination  only  when 
the  solution  is  exceedingly  dilute.  While  these  facts  must  be  interpreted 
to  mean  that  the  copper  salt  tends  to  upset  the  protoplasmic  system  in  some 
way  and  that  it  is  possible  for  that  system  to  be  so  disturbed  as  either  to 
inhibit  germination  absolutely  or  to  allow  this  process  to  proceed  in  modi- 
fied form,  yet  much  more  direct  evidence  of  such  protoplasmic  disturbance 
was  frequently  met  with  in  the  progress  of  this  work.  Spores  in  which 
all  germinational  activity  had  been  prevented  by  the  presence  of  Cu(NO3)2 
frequently  possessed  a  characteristic,  coarsely  granular  appearance,  as 
though  a  precipitate  or  coagulum  had  been  formed  within  the  protoplasm. 
Furthermore,  this  same  appearance  was  often  encountered  in  spores  which 
exhibited  modified  germination  under  the  influence  of  copper.  From  this 
it  appears  that  the  granular  appearance  of  the  protoplasm  does  not  neces- 
sarily denote  death,  but  to  settle  this  point  conclusively  and  to  determine 
whether  the  production  of  apparent  granulation  might  not  be  a  post  mortem 
effect,  the  following  experiment  was  performed. 

Spores  which  had  been  for  eighteen  hours  in  cultures  with  0.00008111 
solution  of  Cu(NO3)2  alone  were  employed.  In  this  solution  no  germina- 
tion has  ever  been  found,  in  the  more  than  twenty  separate  cultures  which 


TOXICITY  OF  HEAVY  METALS  75 

have  been  observed,  and  the  protoplasm  usually  appears  markedly  granular. 
The  spores  were  transferred  from  the  poison  solution  to  a  new  drop  cul- 
ture with  distilled  water  and  were  then  returned  to  the  thermostat.  At 
the  end  of  twenty-four  hours,  observation  showed  that  many  of  the  gran- 
ular spores  had  germinated  normally,  although  the  granular  appearance  was 
still  very  noticeable.  Further  proof  that  the  granulated  appearance  with 
which  we  have  here  to  deal  is  not  a  post  mortem  effect  of  copper  poisoning 
may  be  derived  from  the  fact  that  the  germinal  tubes  of  spores  which 
had  germinated  in  a  combination  solution  of  o.oooim  Cu(NO3),  and 
o.oooo6m  Ca(NO3)2  elongated  considerably  when  the  cultures  were  allowed 
to  remain  in  the  thermostat  for  a  second  day,  although  the  spores  showed 
the  characteristic  granulation  above  mentioned. 

It  seems  probable,  from  these  and  other  similar  lines  of  evidence,  that 
the  copper  of  the  solution  penetrates  the  spore  wall  and  gives  rise  to  the 
precipitation  or  coagulation  effect  just  described. 

It  may  be  noted  here  that  the  influence  of  calcium  upon  the  toxicity  of 
copper  is  of  interest  in  connection  with  the  problem  of  fungicides  and  fungi- 
cidal  action.  From  the  results  obtained  in  the  experiments  described  above, 
the  conclusion  seems  warranted  that  only  a  small  portion  of  the  soluble 
copper  in  Bordeaux  mixture  is  effective  as  a  fungicide.  Yet  it  is  a  well 
known  fact  that  a  long  list  of  fungous  diseases,  including  apple  bitter-rot, 
can  be  controlled  by  the  proper  applications  of  this  mixture  to  the  host 
plants.  The  use  of  KOH  instead  of  Ca(OH)2  in  preparing  the  fungicide 
should  result  in  a  much  more  toxic  mixture,  for,  as  brought  out  in  the 
preceding  experiments,  potassium  is  only  about  one  fourth  as  effective  as 
calcium  in  decreasing  the  toxicity  of  copper.  From  the  standpoint  of 
the  host,  however,  it  is  quite  probable  that  this  anti-toxic  effect  of  the 
calcium  is  important  in  preventing  "  spray-injury  "  to  the  foliage  and  fruit, 
while  the  small  amount  of  soluble  copper  present,  is,  in  most  cases,  suf- 
ficient to  prevent  fungous  growth. 

Lead. 

The  nitrate  of  lead  is  considerably  less  toxic  toward  the  Gloeosporium 
spores  here  employed  than  is  that  of  copper.  The  concentration  just 
inhibiting  germination  after  eighteen  hours  was  found  to  be  about  o.QO4m. 
With  a  concentration  of  0.002111  of  the  lead  salt,  some  of  the  spores  showed 
slight  terminal  or  lateral  swellings.  At  this  concentration  spores  were  fre- 
quently found  in  which  one  half  was  brown  or  blackened  while  the  other 
seemed  devoid  of  protoplasm,  appearing  as  if  the  protoplasmic  contents 
had  all  collected  in  one  end  and  had  then  taken  on  a  thicker  wall.  No 
increase  in  size  or  local  swelling  was  apparent  in  such  cases.  In  a  con- 
centration of  o.ooim  the  blackened  bodies  just  mentioned  were  found,  as 


76  LON  A.  HAWKINS 

well  as  swellings,  and  also  definite  outgrowths  from  one  or  both  ends  of 
the  spore.  The  latter  (shown  in  figure  3)  are  of  particular  interest  and 
require  some  attention  here.  They  are  spherical  in  shape,  dark  brown  or 
black  in  color  and  appear  in  all  respects  similar  to  the  chlamydospores  or 
appressoria  which  have  been  frequently  described  as  resulting  under  certain 
conditions,  from  the  germination  of  spores  of  various  Gloeosporium  forms. 
Appressoria  have  been  described  by  Hasselbring20  as  forming  when 
nutrient  materials  are  absent  and  when  germinating  spores  or  germ  tubes 
come  in  contact  with  such  hard  surfaces  as  are  furnished  by  the  cover 
glass  in  a  drop  culture  or  by  the  epidermis  of  various  fruits.  This  writer 
considers  that  they  function  as  holdfasts  and  that  they  result  from  a  contact 
stimulus  acting  upon  spores  or  tubes  which  are  not  well  nourished.  That 
such  bodies  are  frequently  formed  when  germ  tubes  come  in  contact  with 
the  cover  glass  of  a  hanging  drop  culture,  has  often  been  demonstrated 
in  the  present  studies.  They  are  especially  characteristic  of  cultures  with 
certain  salt  solutions,  at  concentrations  somewhat  below  that  at  which 

germination  is  entirely  suppressed  but  above 
that  at  which  normal  development  occurs.  Here 
their  formation  does  not  appear  to  be  related  to 
any  contact  stimulus,  however.  In  some  cases 

swollen    bodies    are    produced    which    have    the 
Fig.  3.  Germinating       form     Q£     appressoria     but     which     are     haline 
spores       from        o. 00033^1 

Pb(NO )     X6so  ^*e   t"e   usual    spores    and   germ-tubes   of   this 

.fungus,     thus     apparently     differing    from     the 

appressoria  only  in  not  being  brown  or  black  in  color.  In  the  descriptions 
which  follow  the  term  appressoria  will  be  used  to  denote  the  dark  colored, 
appressorium-like  bodies,  and  swellings  of  similar  form  but  without  dark 
appearance  will  be  termed  hyaline  appressoria.  These  terms  are  applied 
here  merely  in  a  descriptive  way,  without  intended  implication  that  the 
bodies  thus  designated  may  not  be  physiologically  or  otherwise  different 
from  the  appressoria  of  the  mycologists. 

In  concentrations  of  Pb(NO3)2  from  o.oooim  to  0.00005111,  most  of  the 
germination  observed  resulted  in  appressoria.  Some  of  the  similar,  hyaline 
swellings  also  occurred  in  these  cultures.  When  cultures  where  appressoria 
were  common  were  allowed  to  remain  in  the  thermostat  for  several  days 
and  were  examined  from  time  to  time,  these  swellings  continued  to  develop 
successively  from  the  same  spore,  until  sometimes  as  many  as  four 
appeared  together  at  one  end  (see  fig.  4).  In  the  formation  of  chains 
of  these  bodies  it  appeared,  from  the  observation  of  different  stages,  that 
a  second  or  later  swelling  may  be  brought  about  either  by  the  germination 
of  one  previously  formed  or  by  enlargement  of  the  constricted  portion  of 

20  Hasselbring,   H.,  The  appressoria  of  anthracnoses.     Bot.  Gaz.  42:   135-142.      1906 


TOXICITY  OF  HEAVY  METALS 


77 


the  tube  between  one  previously  formed  and  the  spore  itself;  in  the  latter 
case  possibly  by  new  growth  from  the  spore.  In  many  cases  a  light  colored 
area  quite  like  that  considered  by  Hasselbring  to  be  a  pore,  could  be  dis- 
tinguished, and  this  may  have  been  present  in  all  cases.  Spores  which  had 
germinated  in  drop  cultures  by  forming  appressoria  were  transferred  to 
new  cultures  in  distilled  water  and  returned  to  the  thermostat.  Nearly 
all  the  appressoria  had  germinated  with  normal  germ  tubes  at  the  end  of 
twenty-four  hours. 

An  occasional  spore  germinated  normally  in  a  concentration  of  0.00005111 
Pb(NO3),,  and  a  few  normal  tubes  were  found  in  the  0.00004111  concen- 
tration, but  it  was  only  in  the  o.oooo2m  concentration  that  no  effect  of  the 
Pb(XO3)2  upon  the  germination  of  the  spores  was  noticeable.  In  the 
latter  concentration  the  spores  germinated  with  long  tubes,  just  as  in  simul- 
taneous controls  in  distilled  water. 

Series  including  combinations  of  Pb- 
(NO3)2  with  Ca(NO3)2  were  carried  out 
at  three  different  concentrations  of  the  lead 
salt,  o.oooi6m,  .0.00033111,  and  o.ooo66m. 
With  a  o.ooo66m  concentration  of  Pb- 
(NO3)2  no  concentration  of  the  calcium  salt 
was  found  which  entirely  suppressed  the 
formation  of  appressoria  and  in  which  only 
normal  germination  was  produced,  though 
with  a  concentration  of  0.05111  Ca(NO3)2 
only  a  few  appressoria  were  found.  This 
combination  gave  practically  the  same  re- 
sults as  were  found  with  a  0.00004111  con- 
centration of  Pb(NO3)2  alone;  i.e.,  some- 
what over  50  per  cent,  of  the  germination  was  normal,  though  a  considerable 
number  of  swellings  and  appressoria  were  formed.  In  the  combination  of 
the  same  concentration  of  Pb(NO3)2  (o.ooo66m)  with  o.oo4m  of  the  cal- 
cium salt  the  effect  produced  was  similar  to  that  found  in  cultures  with 
0.00005111  concentration  of  Pb(NO3)2;  only  occasional  normal  tubes  were 
found,  these  probably  constituting  less  than  I  per  cent,  of  the  total  germina- 
tion. With  concentrations  of  o.ooim,  0.002111  and  0.003111  Ca(NO3)2,  com- 
bined with  a  o.ooo66m  concentration  of  the  lead  salt,  practically  no  normal 
tubes  were  produced,  germination  resulting  in  appressoria  and  swellings. 
These  concentrations  correspond  to  solutions  of  Pb(NO3)2  alone  varying 
in  concentration  from  o.ooim  to  o.oooim. 

In  the  second  series  of  combinations  of  Pb(NO3)2  with  Ca(NO3)2,  the 
concentration  of  the  first  salt  was  o.ooo33m  and  the  same  concentrations  of 
Ca(NO3)2  were  employed  as  in  the  series  just  described.  The  results  in 


Fig.  4.     Spores  after  four  day 
in  o.oo4m   Pb(NO3)2,    X650. 


78  LON  A.  HAWKINS 

these  experiments  were  similar  to  those  of  the  previous  series,  with  some 
differences  due  to  the  lower  concentration  of  the  lead  salt.  Practically  no 
appressoria  were  formed  with  the  combination  containing  the  o.o5m  con- 
centration of  Ca(NO3)2,  germination  being  here  (see  fig.  5)  about  the 
same  as  that  which  occurred  with  the  o.oooo2m  concentration  of  the  lead 
salt  alone.  With  a  combination  containing  o.oim  Ca(NO3)2,  the  same 
effect  was  obtained  as  with  0.00004111  Pb(NO3)2  alone,  and  with  combina- 
tion containing  o.oo2m  Ca(NO3)2  about  the  same  effect  was  evident  as 
with  a  concentration  of  o.oooo5m  Pb(NO3)2  alone.  In  the  combinations 
with  o.ooim  and  0.0005111  Ca(NO3)2  the  results  were  practically  the  same 
as  with  a  concentration  of  o.oooim  Pb(NO3)2  alone;  most  of  the  germina- 
tion here  gave  appressoria. 

With  a  o.oooi6m  concentration  of  Pb(NO3)2  in  combination  with  the 
same  concentrations  of  Ca(NO3)2  as  were  employed  in   the  two  series 


Fig.  5.  Germinating  spores  from  combination 
solution  containing  0.00033111  Pb(NO3)2  and  0.05111 
Ca(NO3)2  X  650. 

above  described,  the  concentration  of  the  calcium  salt  in  which  the  majority 
of  the  germinating  spores  formed  normal  tubes  (instead  of  swellings  and 
appressoria),  and  the  concentration  in  which  only  an  occasional  spore 
formed  a  normal  germ-tube,  were  somewhat  lower  than  in  the  previous 
series,  as  was  to  be  expected.  The  combination  containing  a  o.oo2m  con- 
centration of  Ca(NO3)2  gave  practically  the  same  effect  as  did  the  con- 
centration of  o.oooo/mi  of  the  lead  salt  alone,  while  that  containing  a  o.ooim 
concentration  of  Ca(NO3)2  gave  results  similar  to  those  obtained  with  a 
o.oooo2m  concentration  of  the  lead  salt  alone.  Combinations  containing 
Ca(NO8)2  in  concentrations  of  o.O333m,  0,04111  and  0.05111,  respectively,  pro- 
duced the  same  sort  of  germination  as  was  obtained  with  a  concentration 
of  0.00033111  Pb(NO3)2  combined  with  a  0.05111  concentration  of  the  calcium 
salt ;  germination  was  practically  all  normal,  only  an  occasional  appressorium 
or  swelling  being  found. 

From  the  above   data  the   conclusion   seems   warranted  that  the   same 
molecular  ratio  of  the  calcium  salt  to  that  of  lead  produces  practically 


TOXICITY  OF  HEAVY  METALS  79 

identical  effects  with  the  three  different  concentrations  of  Pb(NO3)2  here 
used.  Concentrations  of  o.oim,  o.O2m  and  o.o6m  Ca(NO3)2  combined, 
respectively,  with  concentrations  of  o.oooi6m,  o.ooo33m  and  o.ooo66m 
Pb(NO3)2  produce  practically  the  same  effect  on  spore  germination  as  does 
the  0.00005111  concentration  of  the  lead  salt  alone.  It  thus  appears  that 
for  all  three  very  different  concentrations  of  Pb(NO3)2,  the  addition  of 
Ca(NO3)2  in  the  proportion  of  six  molecules  of  the  calcium  salt  to  one 
molecule  of  Pb(NO3)2  reduces  the  toxicity  of  the  latter  salt  to  a  point 
where  it  almost,  but  not  quite,  inhibits  normal  germination  and  nearly 
restricts  germinal  activity  to  the  formation  of  appressoria  and  other  swell- 
ings. To  obtain  this  same  condition  of  germination  a  simple  solution  of 
Pb(NO3)2  must  have  a  concentration  of  0.00005111. 

A  similar  series  of  equivalent  effects  upon  spore  germination,  with  the 
three  different  concentrations  of  the  lead  salt,  is  shown  by  taking  as  the 
index  of  physiological  activity  the  prevalence  of  normal  germ  tubes  in 
the  cultures,  as  contrasted  with  swellings  and  appressoria.  This  condi- 
tion of  affairs  is  attained  in  combination  solutions  containing,  respectively, 
o.02m  Ca(NO3)2  combined  with  o.ooo66m  Pb(NO3)2,  o.oim  Ca(NO3)2 
combined  with  0.00033111  Pb(NO3)2,  and  0.005111  (Ca(NO3)2  combined 
with  o.oooi6m  Pb(NO3)2.  These  three  combinations  of  the  two  salts  all 
produce  practically  the  same  effect  on  germination  as  does  the  concentration 
of  0.00004111  Pb(NO3)2  alone.  Here  the  addition  of  about  30  molecules 
of  Ca(NO3)2  to  i  of  Pb(NO3)2  (30:1)  reduces  the  toxicity  of  the  lead 
salt  until  it  is  physiologically  equivalent  to  that  of  the  0.00004111  solution 
of  this  salt  alone. 

Combination  of  Pb(NO3)2  with  Mg(NO3)2  shows  a  similar  influence 
of  the  alkaline  earth  upon  the  toxicity  of  the  heavy  metal,  though  with 
somewhat  different  concentrations.  Equivalent  physiological  effects  were 
produced  by  o.ooo66m  Pb(NO3)2  in  combination  with  o.O2tn  Mg(NO3)2, 
by  0.00033111  Pb(NO3)2  in  combination  with  o.oim  Mg(NO3)2  and  by 
o.oooo5m  Pb(NO3)2  alone.  Here  the  addition  of  about  30  molecules  of 
Mg(NO3)2  for  each  molecule  of  Pb(NO3)2  in  the  solution,  reduced  the 
toxicity  of  the  latter  salt  so  that  it  became  equivalent,  in  both  combinations, 
to  a  simple  solution  containing  0.00005111  Pb(NO3)2.  Under  these  condi- 
tions most  of  the  germinal  activity  took  the  form  of  the  production  of 
appressoria  and  other  swellings,  but  a  small  amount  of  normal  germina- 
tion was  observed  in  all  cases.  Thus,  Mg(NO3)2  appears  to  be  only  about 
one  fifth  as  effective  in  reducing  the  toxicity  of  the  lead  salt  as  is  Ca(NO3)2. 

Magnesium  nitrate  was  not  employed  in  concentration  sufficiently  high 
to  inhibit  all  toxic  effects  of  the  lead  salt.  The  nearest  approach  to  normal 
germination  was  obtained  in  a  combination  solution  containing  o.O5m 
Mg(NO3)o  and  0.00033111  Pb(NO3)2,  in  which  about  half  of  the  germina- 


8o 


LON  A.  HAWKINS 


tion  was  normal.     The  main  points  brought  out  by  these  experiments  are 
summarized  in  table  II. 

TABLE  II 


Type  of  germination 

Physiologically     equivalent     solutions     containing 
Pb(NO3)2     alone     and     in     combination     with 
Ca(NO3)2  or  with   Mg(NO3)2,   molecular 

Molecular 
ratio  in 
combination 
Ca  :Pb  or 
Mg:Pb 

Simple 
.  solutions, 
Pb(N03)2 

Combination  solutions  containing 

Pb(NO3)2 

Ca(NO3)2 

Mg(N03)2 

Mainly  normal.  Some  appres- 
soria  and  other  swellings  .  . 

o  .00004 

o  .00016 
0.00033 
o  .00066 

o  .005 

0  .01 
0  .02 

3°: 
3°: 
30: 

Almost  no  normal  germina- 
tion, mainly  appressoria 
and  other  swellings  

o  .00005 

o  .00016 
0.00033 
0.00033 
o  .00066 
o  .00066 

O  .OI 

o  .02 

o  .01 

0  .02 

6: 
6: 

3°: 
6: 

3°: 

o  .06 

From  the  results  obtained  with  the  different  combinations  of  calcium 
and  magnesium  nitrates  with  Pb(NO3)2,  it  is  evident  that  the  decrease  in 
toxicity  of  Pb(NO3)2,  due  to  the  other  salt,  cannot  be  caused  by  a  depression 
of  the  ionization  of  the  lead  salt  on  account  of  the  common  anion.  In  the 
combination  containing  o.oo4m  Ca(NO3)2  and  o.ooo66m  Pb(NO3)2,  about 
90  per  cent,  of  the  lead  salt  was  calculated  to  be  in  the  dissociated  condi- 
tion. In  the  combination  containing  o.O2m  Ca(NO3)2  or  Mg(NO3)2  to- 
gether with  a  o.ooo66m  concentration  of  the  lead  salt,  the  latter  should  be 
about  40  per  cent,  dissociated.  The  presence  of  either  of  these  concentra- 
tions of  dissociated  lead  salt  alone  in  a  culture  solution  would  either  prevent 
germination  entirely  or  give  only  abnormal  growth.  That  the  decrease  in 
toxicity  may  have  been  due  to  the  formation  of  a  double  salt  remains 
possible ;  at  least  no  direct  evidence  to  the  contrary  was  obtained. 

With  the  concentrations  of  Pb(NO3)2  that  inhibited  germination  no 
granular  appearance  of  the  protoplasm,  such  as  was  found  in  otherwise 
physiologically  similar  solutions  of  Cu(NO3)2,  was  evident.  Yet  it  is 
clear  that  the  lead  salt  either  directly  or  indirectly  affects  the  protoplasm 
through  the  spore  wall,  as  is  shown  by  the  formation  of  the  dark  bodies 
occupying  one  half  of  the  spore  in  many  cases.  This  response  was  observed, 
as  has  been  mentioned,  in  the  higher  concentrations  of  the  lead  salt.  A 
general  discussion  of  the  different  forms  of  germination  will  be  taken  up 
below,  after  the  effects  of  the  combinations  of  calcium  and  magnesium 
nitrates  with  the  other  salts  have  been  presented. 


TOXICITY  OF  HEAVY  METALS  81 

Zinc. 

Zinc  nitrate,  alone  and  in  combination  with  the  nitrates  of  calcium  and 
magnesium,  was  studied  in  much  the  same  way  as  were  the  nitrates  of 
copper  and  lead.  Zinc  nitrate  alone  inhibited  germination  in  o.25m  concen- 
tration. Many  local  swellings  of  the  spore  walls  were  found  in  concen- 
trations of  o.oSm  and  0.04111,  while  local  swellings  and  short  tubes  were 
present  in  concentrations  of  0.008111  and  0.004111.  Normal  germination 
occurred  with  the  concentration  of  o.oo2m.  Combinations  of  Zn(NO3)2 
with  Ca(NO3)2  and  Mg(NO3)2  were  tested,  using  a  concentration  of 
0.04111  Zn(NO3)2.  In  these  combinations  the  calcium  salt  was  employed 
in  concentrations  ranging  from  0.000125111  to  o.O2m.  Some  normal  germ- 
ination occurred  with  all  concentrations  of  Ca(NO3)2  below  and  including 
that  of  0.0005111,  but  none  was  observed  in  the  mixture  containing  a 
o.oooi25m  concentration  of  this  salt.  It  thus  appears  that,  normal  germ- 
ination of  these  spores  may  be  brought  about  in  a  0.04111  Zn(NO3)2  solu- 
tion, by  the  addition  of  Ca(NO3)2  in  the  proportion  of  one  molecule  of  this 
to  every  80  molecules  of  Zn(NO3)2  present. 

The  concentrations  of  Mg(NO3)2  which  were  employed  in  these  com- 
binations with  the  0.04111  solution  of  Zn(NO3)2  ranged  from  0.00025111  to 
0.025111.  Here  it  was  found  that  no  normal  germination  occurred  until  the 
magnesium  salt  reached  a  concentration  in  the  mixture  of  0.0025111.  This 
means  that  to  produce  any  normal  germination  in  the  Zn(NO3),  solution 
here  used,  by  addition  thereto  of  Mg(NO3),,  it  is  necessary  to  add  one 
molecule  of  the  latter  salt  for  every  16  molecules  of  Zn(NO3)2  already 
present.  It  therefore  requires  five  times  as  much  of  the  magnesium  salt 
to  counteract  the  toxic  influence  of  Zn(NO3)2  in  the  concentration  here 
used  (0.04111)  as  is  required  of  the  calcium  salt  to  produce  the  same  effect. 

The  relatively  small  amounts  of  the  calcium  and  magnesium  salts,  which 
are  required  to  inhibit  the  toxic  effects  of  Zn(NO3)2  in  0.04111  concentra- 
tion, preclude  any  possibility  that  the  antagonistic  influence  of  either  of  the 
former  salts  might  be  related  to  decreased  dissociation  of  the  zinc  salt, 
brought  about  by  their  addition,  so  that  this  consideration  needs  no  atten- 
tion in  this  case. 

It  is  interesting  to  recall  here  that  the  effectiveness  of  Mg(NO3)2  in 
counteracting  the  toxicity  of  Pb(NO3)2,  in  the  two  concentrations  studied, 
so  as  to  allow  some  normal  germination  of  the  spores,  was  also  found  to 
be  only  about  one  fifth  as  great  as  that  of  Ca(NO3)2.  The  relative  effec- 
tiveness of  the  nitrates  of  calcium  and  magnesium  thus  seems  to  be  the 
same  whether  they  are  employed  to  counteract  the  toxicity  of  Pb(NO3)2 
or  that  of  Zn(NO3)2. 

Zinc  nitrate  did  not  seem  to  stimulate  the  spores  to  form  appressoria, 
though  hyaline  swellings  were  common  in  the  more  concentrated  solutions. 


82 


LON  A.  HAWKINS 


No  direct  evidence  was  obtained  as  to  whether  or  not  zinc  nitrate  enters 
the  spores. 

Aluminum. 

Aluminum  nitrate  was  used  at  certain  concentrations,  both  alone  and  in 
combination  with  magnesium  and  calcium  nitrates.  Series  were  carried 
out,  and  several  times  repeated,  including  the  combinations  and  concentra- 
tions which  are  presented  in  table  III. 


TABLE  III 


Concentration  of  Al(NO3)3 
molecular 


Concentration  of  Mg(NO,)2 

or  of  Ca(NO3)2, 

molecular 


Common 
fraction 

Decimal 
fraction 

Common 
fraction 

Decimal 
fraction 

9/2000 

0.0045 

I/IOO 

0  .01 

8/2OOO 

o  .004 

2/IOO 

o  .02 

6/2OOO 

0.003 

4/100 

o  .04 

4/2000 

o  .002 

6/100 

o  .06 

3/2000 

o  .0015 

7/100 

o  .07 

2/2000 

0  .001 

8/100 

0.08 

1/2000 

o  .0005 

9/100 

o  .09 

1/4000 

o  .00025 

95/1000 

0.095 

In  none  of  the  cultures  with  A1(NO3)3  did  the  addition  of  either 
Mg(NO3)2  or  Ca(NO3)2  bring  about  any  alteration  at  all  in  the  behavior 
of  the  spores.  Although  many  other  proportions  of  these  pairs  of  salts 
might  have  been  tried,  it  appears  probable  that,  if  either  the  calcium  or 
magnesium  salt,  at  any  concentration,  exerts  any  influence  upon  the  toxicity 
of  A1(NO3)3,  in  any  concentration,  some  evidence  in  this  direction  would 
have  been  obtained  from  the  experiments  which  were  carried  out.  This 
seems  still  more  probable  from  the  fact  that  different  concentrations  of 
the  aluminum  salt  alone  produced  markedly  different  effects  upon  the 
activities  of  the  spores,  a  matter  which  will  now  receive  attention. 

In  the  highest  concentration  of  A1(NO3)3  used  in  the  combinations, 
namely  0.0045111,  only  a  few  local  swellings  of  the  spores  were  observed. 
A  blackening  of  half  of  the  spore  was  frequently  found  here  (fig.  6).  No 
normal  germination  occurred  in  this  solution.  In  the  two  lowest  concen- 
trations of  A1(NO3)3  (0.005111  and  0.00025111)  however,  much  of  the  germ- 
ination was  normal.  Aluminum  nitrate  appears  to  influence  the  form  of 
germination  occurring  in  these  spores,  with  much  the  same  results  as  have 
been  described  for  cultures  poisoned  with  Pb(NO3)2.  The  types  of  renewed 


TOXICITY  OF  HEAVY  METALS  83 

activity  which  occur  with  A1(NO3)3  are:  (i)  the  formation  of  a  dark  body 
occupying  about  half  of  the  spore,  (2)  the  formation  of  a  small  local 
swelling  at  one  end  of  the  spore,  and  (3)  the  development  of  one  or  more 
appressoria,  the  latter  sometimes  having  their  characteristic  dark  color  and 
sometimes  being  hyaline.  Unless  the  formation  of  the  internal  dark  bodies, 
which  seem  to  arise  directly  through  protoplasmic  activity,  may  be  taken  as 
evidence  of  a  penetration  of  the  toxic  salt,  no  information  as  to  whether 
A1(NO3)3  actually  enters  the  spores  was  obtained. 


Nickel. 

Nickel  nitrate  proved  to  be  very  slightly  toxic  to  these  fungus  spores,  its 
effect  on  germination  being  manifest  only  in  very  high  concentrations.  On 
this  account  no  study  was  made  of  the  possible  influence  of  calcium  and 
magnesium  nitrates  upon  this  toxicity. 

The  toxic  effects  of   Ni(NO3)2 
alone  may  be  added  here.     A  con- 
centration  of   o.5m   inhibited   ger- 
mination.     In    a    concentration    of 
0.25111,  numerous  local  swellings  of 
the   spores    were   observed.      Both 
local  swellings  and  normal  germina- 
tion   were    present    in    a    0.12501 
solution  of  this  salt,  and  germina- 
tion  was    perfectly   normal,    as    in 
the   distilled   water  control,   where 
a  concentration  of  o.o5m  or  lower  was  employed.     Germinating  spores  in 
o.25m  concentration  of  Ni(NO3)2  frequently  exhibited  a  granular  appear- 
ance such  as  has  been  described  for  spores  in  solutions  of  Cu(NO3)2  which 
were  not  quite  concentrated  enough  to  inhibit  all  activity. 


Fig.  6.     Germinating    spores 
o.oo2m  Al   (NO3)3,    X    650. 


from 


TOXICITY    OF    SINGLE    SALTS 

The  present  section  will  be  devoted  to  the  relative  degrees  of  toxicity 
exhibited  by  the  various  substances  heretofore  dealt  with,  together  with 
certain  other  considerations  bearing  upon  the  problem  of  chemical  stimu- 
lation as  evidenced  in  the  simple  solutions  employed  in  these  studies.  Besides 
the  substances  already  mentioned,  nitric  acid  entered  into  the  experimen- 
tation, and  the  information  gained  regarding  the  toxicity  of  this  substance 
will  also  find  place  here. 

From  what  has  preceded  it  appears  possible  to  group  the  effects  of  the 
different  treatments  into  four  distinct  kinds  of  physiological  response  on 
the  part  of  the  fungus  spores  here  employed,  (i)  a  given  treatment  may 


84  LON  A.  HAWKINS 

be  without  apparent  effect;  under  such  treatments  the  spores  germinate 
normally  within  eighteen  hours,  just  as  they  do  in  the  control  with  dis- 
tilled water.  (2)  Germinal  activity  may  be  manifest,  but  in  other  ways 
than  that  which  is  here  considered  as  normal.  Under  treatments  which 
produce  this  sort  of  response,  the  activities  of  the  spores  result  sometimes 
(a)  in  an  actual  decrease  in  the  exposed  surface  as  well  as  in  the  volume 
of  the  organism,  and  sometimes  (b)  in  an  increase  in  both  volume  and 
surface,  the  latter,  however,  increasing  much  less  markedly, — as  related  to 
increase  in  volume, — than  is  the  case  in  normal  germination.  In  the  first 
category  (a,  above)  belong  the  phenomena  involved  in  the  production  of 
the  dark  bodies  which  have  been  described  as  forming  within  the  spore  wall 
and  occupying  only  a  portion  of  its  volume.  These  appear  to  have  essen- 
tially the  characteristics  of  chlamydospores  formed  within  the  organism. 
All  other  renewed  activities  of  the  spores  which  have  been  described  belong 
to  the  second  category  (b),  and  embrace  those  growth  processes  which 
result  in  more  or  less  restricted  swellings,  especially  at  the  ends  of  the 
spores,  in  appressoria,  with  either  dark  or  hyaline  wall,  and  in  short  germ 
tubes  somewhat  resembling  the  normal  tubes  but  of  much  greater  diameter. 
(3)  No  renewed  activity  may  occur  at  all  during  a  period  of  from  eighteen 
to  twenty  hours  under  the  given  treatment,  although  ability  to  germinate 
if  transferred  from  this  treatment  to  another  (as  to  distilled  water)  may 
still  be  retained.  Here  the  treatment  prevents  germinal  activity  but  does 
not  destroy  viability,  at  least  within  the  given  time  limits.  (4)  Viability, 
or  power  to  germinate  in  water,  may  be  destroyed  within  a  period  of  from 
eighteen  to  twenty-four  hours;  the  organism  is  killed  outright. 

It  has  already  become  clear  in  this  work  that  most  of  the  substances 
dealt  with  produce  death  within  the  assumed  time  limits,  if  applied  at  a 
sufficiently  high  concentration  (4).  With  a  somewhat  lower  concentration 
of  the  injurious  material,  germination  is  inhibited  but  viability  is  retained 
throughout  the  given  period  (3).  When  the  concentration  is  still  lower 
germinal  activity  becomes  manifest  but  takes  other  forms  than  those  recog- 
nized as  normal  (2).  Finally,  when  the  concentration  of  the  toxic  agent 
is  still  further  decreased,  the  stimulation  threshold  in  the  present  sense  is 
passed  and  normal  germination  becomes  the  rule  (i). 

Livingston21  working  with  a  green  alga  has  presented  a  somewhat  similar 
series  of  responses  to  chemical  stimuli.  This  writer  studied  the  effect 
of  a  large  number  of  nitrates  and  sulphates  on  a  form  of  Stigeoclonium, 
adding  different  concentrations  of  the  salts  to  a  nutrient  medium  in  which 
the  alga  was  grown.  With  a  dilute  nutrient  solution,  in  which  the  alga 
had  a  characteristic  filamentous  form,  its  response  to  stimulation  might  be 
considered  as  of  three  types,  (i)  death,  (2)  change  in  phenomena  of 

21  Livingston,  B.E.,  Chemical  stimulation  of  a  green  alga.     Bull.  Torr.  Bot.  Club  32:  1-34.     1905. 


TOXICITY  OF  HEAVY  METALS  85 


growth,  and  (3)  change  in  phenomena  of  reproduction.  The  response  to  a 
high  concentration  was  usually  death,  while  addition  of  a  toxic  salt  in  con- 
centration somewhat  below  the  fatal  one  stimulated  the  production  of  the 
palmella  form,  with  spherical  cells  and  division  occurring  in  all  directions. 
It  is  interesting  that,  with  relatively  similar  concentrations  of  the  salts, 
(that  is  at  concentrations  somewhat  lower  than  those  required  to 
inhibit  germination)  the  spores  dealt  with  in  the  present  paper  exhibited 
responses  similar  to  those  which  Livingston  found  with  his  alga ;  the  pro- 
duction of  appressoria  and  swellings  in  this  fungus  appears  physiologically 
similar  to  the  production  of  the  palmella  form  in  Stigeoclonium.  The  third 
type  of  response  discussed  by  Livingston,  change  in  phenomena  of  repro- 
duction, of  course  finds  no  parallel  m  this  investigation. 

For  convenience  of  comparison,  the  limits  of  the  various  responses  of 
the  fungus  spores  here  dealt  with,  to  the  different  treatments  employed, 
are  presented  in  table  IV.  The  only  nitrate  occurring  in  the  table  that  has 
not  already  received  attention  is  the  acid.  In  a  series  of  cultures  with 
HNO3  the  germination  at  a  0.005111  concentration  was  practically  all  normal, 
while  at  o.oim  and  all  higher  concentrations,  no  germination  took  place  in 
the  eighteen  hour  period.  The  acid  prevented  germination  for  the  period 
of  eighteen  hours,  in  a  o.O2m  concentration  and  killed  the  spores  in  0.5111. 
No  concentration  was  found,  therefore,  at  which  the  germination  took  the 
form  of  any  of  the  various  abnormal  growths  found  at  some  concentration 
with  all  the  other  substances  used,  nor  was  there  any  apparent  coagulation 
of  the  protoplasm  at  concentrations  below  that  which  killed  the  spores. 
From  these  considerations  it  seems,  then,  that  the  various  abnormal 
growths  and  effects  on  the  protoplasm  of  the  spore  cannot  be  due  to 
acid  present  in  the  salt  solutions,  as  a  result  of  hydrolysis  of  the  salts, 
but  must  be  related,  either  directly  or  indirectly,  to  the  metals  themselves. 

Turning  now  to  table  IV,  the  different  substances  are  there  arranged  in 
the  order  of  the  concentration  which  just  allowed  normal  germination  in  a 
period  of  eighteen  hours.  In  the  first  column  are  listed  the  substances 
dealt  with.  The  remaining  columns  are  each  double.  The  second  column 
presents  the  concentrations  in  which  the  spores  are  killed  in  eighteen  hours 
and  fail  to  germinate  later,  after  transfer  to  distilled  water.  Here,  as  in 
many  other  instances,  the  critical  concentrations  must  not  be  regarded  as 
definite  in  the  sense  of  the  more  exact  physical  sciences ;  the  concentrations 
employed  in  the  experimental  series  were  frequently  rather  widely  separated, 
and.  were  this  not  the  case,  the  variability  of  the  organism  in  its  resistance 
to  toxic  substances  would  render  quite  useless  any  attempt  to  define  such 
critical  points  with  very  great  accuracy.  In  all  such  work  as  the  present, 
dealing  with  organisms,  the  internal  conditions  of  the  cells  must  be  as 
important  in  determining  reactions  as  are  the  external  ones,  and  we  are 


86 


LON  A.  HAWKINS 


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TOXICITY  OF  HEAVY  METALS  87 

as  yet  unable  either  to  control  or  measure  the  former,  excepting  in  a  very 
general  way. 

In  the  third  column  are  found  the  concentrations  at  which  the  different 
salts  inhibit  germination  for  at  least  eighteen  hours.  When  spores  from 
these  cultures  were  removed  from  the  toxic  solution  and  placed  in  water 
for  a  day,  they  germinated.  The  fourth  column  gives  the  maximum  con- 
centration in  which  germination  occurred.  In  all  cases  this  growth  was 
abnormal,  showing  swellings,  swollen  tubes  and  other  unusual  structures, 
such  as  are  illustrated  in  figs,  i  to  6.  In  the  fifth  column  the  concentrations 
given  are  the  maxima  in  which  any  normal  germination  was  observed.  The 
sixth  column  presents  the  highest  concentrations  in  which  no  toxic  influ- 
ence was  manifest. 

From  table  IV  it  will  be  seen  that  the  copper  salts  are  by  far  the  most 
toxic  of  all  the  salts  here  studied.  These  two  salts  are  very  similar  in 
their  effect  on  the  germination  of  the  spores,  which  is  in  accord  with  the 
results  of  Clark  [99],  and  points  almost  conclusively  to  the  commonly 
accepted  idea  that  the  toxicity  of  such  copper  salts  is  mainly  or  entirely 
due  to  the  copper  ion.  The  last  mentioned  writer  found  that  the  concen- 
tration of  copper  salts  which  inhibited  germination  were  higher  than  those 
producing  the  same  effect  in  this  investigation,  a  fact  that  may  probably 
be  due  to  internal  differences  in  the  organisms  dealt  with;  he  found  that 
his  five  fungi  varied  markedly  in  their  response  to  the  same  stimulus.  Also, 
Clark  usually  made  use  of  nutrient  media,  while  the  present  studies  were 
carried  out  without  their  employment.  It  is  quite  possible  that  the  presence 
of  nutrient  substances  might  have  modified  the  effect  of  the  poison.  Dug- 
gar22  found  the  nitrate  of  copper  considerably  more  toxic  than  the  sulphate 
and  also  considerably  more  so  than  it  is  here  shown  to  be.  In  the  work 
of  Stevens,23  these  two  salts  affected  germination  similarly  and  the  concen- 
trations required  to  prevent  germination  varied,  with  the  different  fungi 
used,  from  111/6400  to  111/200.  Many  other  investigations  of  the  effect  of 
copper  on  the  germination  of  fungus  spores  have  been  carried  out,  with 
varying  results.  The  effect  of  this  poison  on  algae  has  been  studied  by 
Livingston  [05].  Kahlenberg  and  True,24,  Heald,25  Jensen,26  Szucs  [12] 
and  others,  have  investigated  its  toxicity  toward  higher  plants.  The  results 
obtained  indicate  that  fungus  spores  are  considerably  more  resistant  toward 
the  toxic  effects  of  copper  than  are  either  the  algae  or  higher  plants. 


tfar,  B.M.,  Physiological  studies  with  reference  to  the  germination  of  certain  fungus  spores. 
Bot.  Gaz.  31:  38-65.  1901. 

23  Stevens,  F.L.,  The  effect  of  aqueous  solutions  upon  funtjus  spores.     Bot.  Gaz.  26:  377-406.      1898. 

2J  Kahlenberg,  Louis,  and  True,  Rodney  H.,  On  the  toxic  action  of  dissolved  salts  and  their  elec- 
trolytic dissociation.  Bot.  Gaz.  aa:  81-124.  1896. 

25  Heald.  F.D.,  The  toxic  effect  of  dilute  solutions  of  acids  and  salts  upon  plants.  Bot.  Gaz.  22: 
I2S-IS3-  1896. 

28  Jensen,  G.H.,  Toxic  limits  and  stimulation  effects  of  some  salts  and  poisons  on  wheat.  Bot.  Gaz. 
43:  11-44.  1907- 


88  LON  A.  HAWKINS 

In  the  order  of  their  toxicity,  Pb(NO3)2  follows  the  copper  salts,  though 
not  closely,  with  A1(XO3)3  next.  The  effects  of  the  last  two  salts  on 
plants  seems  to  have  been  studied  but  little.  Livingston  [05]  employed 
them,  however,  and  found  the  same  relation  to  hold  true  as  is  here  brought 
out.  Also,  Jensen  [07]  found  that  Pb(NO3)2  is  somewhat  less  toxic  toward 
wheat  seedlings  than  is  Cu(NO3)2.  The  nitrates  of  zinc  and  nickel,  which 
are  next  in  the  order  of  diminishing  toxicity,  are  not  exceedingly  toxic 
toward  these  Gloeosporium  spores.  It  is  interesting  to  note  that  Clark  [99], 
working  with  fungi,  and  Livingston  [05],  with  an  alga,  found  nickel  salts 
to  be  more  strongly  toxic  than  those  of  zinc,  results  which  are  opposite 
to  those  obtained  in  this  investigation. 

Magnesium,  calcium,  and  potassium  nitrates  and  cane  sugar  end  the  list, 
in  the  order  named.  It  is  noticeable  that,  with  these  last  four  compounds 
at  concentrations  just  below  that  required  to  inhibit  germination,  growth 
takes  the  form  of  swollen  bodies  which  tend  to  give  a  lower  value  to  the 
ratio  of  surface  to  volume  of  the  organism  than  that  resulting  from  normal 
germination.  This  reaction  to  toxic  stimuli,  at  concentrations  just  below 
those  required  to  inhibit  growth,  occurs  commonly  with  all  the  salts  here 
used.  That  it  is  found  with  cane  sugar,  which  is  usually  regarded  as 
non-toxic  (see  True27)  suggests  that  such  reactions  may  be  brought  about 
by  high  osmotic  pressure  as  well  as  by  chemical  stimulation  in  the  true 
sense  of  this  term.  This  suggestion  is  in  accord  with  the  conclusions  of 
Livingston  [05],  who  has  shown  that  his  form  of  Stigeoclonium  assumes 
the  palmella  form  in  response  either  to  a  chemical  stimulus  or  to  one  of 
relatively  high  osmotic  pressure.  Whether  this  effect  of  sugar,  and  similar 
effects  produced  by  high  concentrations  of  calcium,  magnesium  and 
potassium  nitrates  is  really  due,  in  the  present  instances,  to  osmotic  pressure 
relations,  was  not  determined  experimentally ;  the  minuteness  of  the  spores 
must  render  experiments  involving  plasmolysis  exceedingly  difficult  with 
such  organisms  as  those  here  employed.  It  is  obvious,  however,  that,  the 
high  concentrations  of  the  calcium,  magnesium  and  potassium  salts,  found 
necessary  to  affect  germination,  must  be  capable  of  exerting  an  extremely 
high  osmotic  pressure  if  the  ectoplasm  of  the  spore  is  not  readily  permeable 
to  them. 

From  table  IV  it  may  be  seen  that,  with  the  exception  of  HNO3,  the  dif- 
ferent compounds  arrange  themselves  in  the  same  order  no  matter  what 
type  of  reaction  to  the  chemical  stimuli  may  be  taken  as  criterion.  This 
suggests  that  the  physiological  response  of  death,  on  the  one  hand,  and 
the  various  morphogenic  responses  considered  in  this  paper,  on  the  other, 
are  ultimately  related  to  the  same  properties  or  characteristics  of  the  various 

27  True,  Rodney  H.,  The  physiological  effects  of  certain  plasmolyzing  agents.     Bot.  Gaz.  26:  407- 
416.     1898. 


TOXICITY  OF  HEAVY  METALS  89 

stimulating  substances  which  bring  them  about.  Copper  4s  the  most  toxic 
metal  here  dealt  with,  whether  we  consider  its  toxicity  as  bringing  about 
changes  that  result  in  death  or  as  bringing  about  the  less  final  changes 
that  lead  to  abnormal  growth.  In  the  same  way,  magnesium,  calcium  and 
potassium  are  the  least  toxic  metals  of  the  list,  on  whatever  physiological 
criterion  we  may  base  our  judgment. 

GENERAL  DISCUSSION  AND  CONCLUSION. 

The  results  of  the  experiments  described  in  the  foregoing  pages  show 
that,  in  certain  cases  at  least,  the  effect  of  a  toxic  salt  on  the  germination 
of  the  conidia  of  Glomerella  cingulata  may  be  influenced  by  the  addition, 
to  the  medium,  of  calcium,  magnesium  or  potassium  nitrate.  That  this 
effect  is  not  due  to  depression  of  ionization  of  the  toxic  salt  has  been 
demonstrated.  That  it  cannot  be  due  to  the  formation  of  undissociated 
double  salts  has  been  shown  for  the  combination  of  Cu(NO3)2  with 
Ca(  NO3)2  and  of  Zn(NO3)2  with  calcium  or  magnesium  nitrate.  The  influ- 
ence of  calcium  upon  the  toxicity  of  the  salts  of  the  heavy  metals  here 
employed  must  then  be  related  to  an  effect  of  the  Ca(NO3)2  on  the  spore, 
or  to  an  effect  on  the  contained  protoplasm,  as  is  made  otherwise  probable 
from  the  fact  that  copper,  lead,  aluminum  and  nickel  nitrates  sensibly  effect 
the  protoplasm  in  various  ways,  without  producing  any  apparent  changes 
in  the  spore  wall. 

There  are,  of  course,  a  number  of  logically  possible  explanations  for  the 
antagonistic  actions  of  salts  upon  the  germination  of  fungus  spores  as 
here  recorded,  but  any  such  possible  hypothesis  must  necessarily  be  «yery 
complex  if  it  be  able  to  account  generally  for  even  the  small  array  of 
facts  so  far  at  our  disposal. 

Various  theories  have  been  suggested  from  time  to  time  to  explain  similar 
salt  antagonisms.  Loeb28  has  advanced  the  theory  of  ion-proteid  formation, 
suggesting  that  one  or  both  ions  of  a  salt  may  combine  with  proteids  of 
the  protoplasm  to  form  ion-proteid  compounds.  This  writer  found,  for 
example,  that  a  solution  of  NaCl  is  poisonous  unless  some  calcium  and 
potassium  be  also  present  in  the  medium,  in  which  case  the  toxicity  of 
NaCl  is  greatly  decreased  or  inhibited  altogether.  He  considers  that  cal- 
cium and  potassium  ions  may  to  some  extent  take  the  place  of  the  sodium 
ion  in  the  ion-proteid  compounds  above  mentioned,  and  suggests  that  the 
organism  cannot  live  without  some  of  these  proteid  compounds  containing 
calcium  and  potassium  as  well  as  those  containing  sodium.  The  work  of 
Osterhout 29  on  balanced  solutions  supports  this  theory  of  Loeb. 

28  Loeb,  J.,  On  ion-proteid  compounds  and  their  role  in  the  mechanics  of  life  phenomena.     I.  The 
poisonous  character  of  a  pure  NaCl  solution.     Amer.  Jour.  Physiol.  7:  327-138.      1900. 

29  Osterhout,  W.  J.  V.  [06],  On  the  importance  of  physiologically  balanced  solutions  for  plants.     II. 
Fresh  water  and  terrestrial  plants.     Bot.  Gaz.   44:  259-272.      1907. 

PHYSIOLOGICAL   RESEARCHES   VOL.    I,    NO.    2,    SERIAL   XO.   2 

AUGUST,    191^.  • 


9O  .     LON  A.  HAWKINS 

Wo.  Ostwald,30  from  his  studies  on  Gammarus,  concludes  that  the  toxicity 
of  a  solution  is  related  to  the  power  of  the  protoplasm  to  adsorb  the  dis- 
solved substance,  saying  "  dass  eine  Losung  um  so  giftiger  istje  starker 
sie  adsorbiert  wird."  Morowitz31  somewhat  similarly  considers  that  the 
toxic  salt  is  adsorbed  by  the  surface  layer  of  the  organism  and  diffuses 
thence  into  the  interior,  the  amount  thus  entering  in  a  given  time  being 
related  to  the  rate  of  adsorption.  He  supposes  that,  if  an  otherwise  indif- 
ferent but  strongly  adsorbed  substance  be  simultaneously  present  in  the 
medium,  the  adsorption  of  the  latter  decreases  the  power  of  the  surface 
layer  to  adsorb  the  toxic  salt,  so  that  a  smaller  amount  of  this  enters  the  cell. 

True  and  Gies  [03]  also  relate  these  antagonistic  effects  to  protoplasmic 
properties,  and  seem  to  consider  the  antagonism  between  copper  and  calcium 
to  be  due  to  an  accelerating  effect  of  calcium  acting  against  a  retarding 
influence  exerted  by  copper.  Sziics  [12],  as  has  already  been  mentioned, 
likewise  supposes  the  influence  of  one  salt  upon  the  toxicity  of  another  to 
be  due  to  simultaneous  effects  of  the  two  salts  upon  the  protoplasm. 

Loeb32  has  rather  recently  advanced  the  theory  that  the  influence  of  one 
salt  on  the  toxic  action  of  another  is  due  to  changes  brought  about  in  the 
outer  colloidal  membrane  of  the  protoplasm,  whereby  the  latter  becomes 
impermeable  to  the  toxic  salt.  Osterhout,33  working  with  cut  disks  of 
Laminaria  and  his  method  of  electrical  conductivity,  also  comes  to  the  con- 
clusion that  ([12],  page  115)"  the  antagonistic  action  of  salts  is  largely  or 
entirely  due  to  the  fact  that  they  hinder  or  prevent  one  another  from 
entering  the  protoplasm." 

It  will  be  seen  that  any  one  of  the  various  hypotheses  just  reviewed  may, 
with  adequate  quantitative  assumptions,  explain  the  phenomena  of  antagon- 
ism as  these  are  exhibited  by  any  particular  pair  of  concentrations  of  any 
particular  pair  of  salts  dealt  with  in  the  present  study.  It  seems,  impos- 
sible, however,  to  formulate  a  hypothesis,  either  by  means  of  any  of  these 
published  suggestions  or  with  any  other  logical  possibilities,  which  may 
simultaneously  and  satisfactorily  account  for  all  the  cases  with  which  we 
have  to  deal.  The  marked  differences,  brought  out  in  the  present  studies, 
between  the  behavior  of  calcium  when  employed  with  copper  and  that  of 
the  same  element  in  combination  with  lead,  zinc  or  aluminum,  strongly 
suggest  the  probability  that  valence  alone  does  not  determine  the  efficiency 
of  one  cation  in  influencing  the  toxicity  of  another.  It  appears  that  other 
properties  must  be  taken  into  account  if  such  differences  are  to  be  explained. 

30Ostwald,  Wo.,  Ueber  die  Beziehungen  zwischen  Adsorption  und  Gifti^keit  von  Salzl<">sum?en  fur 
Susswassertiere  (Gammarus).  Archiv.  Ges.  Physiol.  120:  10-30.  1007. 

31  Morowitz,   Huso,  Ueber  Adsorption  und   Kolloidfalluns;.      Kolloidchem.  Beih.   i:  301-331.      IQIO. 

32  Loeb,  J.,  The  mechanistic  conception  of  life.     Chicago.      1912.     Page    173. 

33  Osterhout,  W.J.V.,  The  permeability  of  protoplasm  to  ions  and  the  theory  of  antagonism.    Science 
N.S.  35:   112-115.     1012. 

.Some  quantitative  researches  on  the  permeability  of  plant  colls.     Plant  World  16  :  T  20-144.     1013- 


TOXICITY  OF  HEAVY  METALS  91 

A  consideration  of  the  toxicities  of  the  single  salts,~aa  brought  out  in 
this  investigation,  also  suggests  that  the  influence  of  each  one  of  these  salts 
upon  the  protoplasm  of  the  spore  is  specific.  The  nitrates  of  lead  and 
aluminum,  in  concentrations  somewhat  below  those  which  inhibit  germina- 
tion, frequently  produce  within  the  spore  (either  directly  or  indirectly)  a 
dark,  chlamydospore-like  body.  The  presence  of  copper,  at  a  similar  con- 
centration as  regards  toxicity,  causes  a  granular  appearance  of  the  proto- 
plasm and  much  the  same  effect  was  observed  in  concentrations  of  nickel 
nitrate  not  quite  strong  enough  to  inhibit  germination.  Toxic  concentra- 
tions of  zinc  nitrate,  however,  fail  to  produce  any  visible  alteration  in  the 
protoplasm.  The  effects  of  the  different  single  salts  upon  the  spores  are 
therefore  not  at  all  the  same,  and  it  seems  at  least  reasonable  to  suppose 
that  the  requisite  antidotes  may  not  be  identical  and  may  not  be  effective 
in  the  same  manner  in  all  cases.  A  given  substance  at  a  given  concentration 
may  inhibit  the  poisonous  effects  of  one  toxic  salt  and  yet  have  no  influ- 
ence upon  the  toxicity  of  another.  Thus,  neither  calcium  nor  magnesium 
nitrate  exerts  any  apparent  influence  upon  the  toxic  effect  of  aluminum 
nitrate,  but  both  are  markedly  effective  in  counteracting  the  toxicity  of  the 
corresponding  salts  of  zinc  and  lead. 

The  conclusion  seems  unavoidable,  therefore,  that  no  simple  and  broadly 
general  explanation  can  be  applied  to  these  exceedingly  various  antagonistic 
actions,  and  that  the  explanation  of  each  particular  case  must  involve 
the  chemical  characteristics  of  the  salts  concerned  and  the  physico-chemical 
properties  of  their  solutions.  The  exceeding  complexity  of  the  material 
system  within  the  cell  must  make  possible  a  great  variety  of  explanations 
until  this  variety  becomes  further  limited  by  increased  knowledge.  As  has 
been  well  emphasized  by  Osterhout  [13]  the  future  advance  of  our  knowl- 
edge of  this  intricate  subject  must  depend  solely  upon  further  investigation 
of  a  quantitative  nature. 

THE  LABORATORY  OF  PLANT  PHYSIOLOGY 
OF  THE  JOHNS  HOPKINS  UNIVERSITY. 
APRIL   i,   1913. 

LITERATURE  CITED. 

Numbers  in  brackets  throughout  the  preceding  pages  refer  to  the  year  of  publi- 
cation and  to  the  corresponding  numbers  which  follow  authors'  names  in  this  list. 

Acree,   S.F.  [n].     See  Loomis  and  Acree   [n]. 

Bartlett.   H.H.  [12].     See  True  and  Bartlett  [12]. 

Clark,  J.F.  [99],  On  the  toxic  effect  of  deleterious  agents  on  the  germination  and  development  of  cer- 
tain filamentous  fungi.     Bot.  Gaz.   28:   280-327,   378-404.      1899. 

[01],  On  the  toxic  value  of  mercuric  chloride  and  its  double  .salts.     Jour.  Physic.  Chem.  5: 

289-316.      1901. 

-     [02],  On  the  toxic  properties  of  some  copper  compounds  wth  special  reference  to  Bordeaux 
mixture.     Bot.  Gaz.    33:   26-48.      1902. 


92  LON  A.  HAWKINS 

Duggar,  B.M.  [bi],  Physiological  studies  with  reference  to  the  germination  of  certain  fungus  spores.  Bot. 
Gaz.  31 :  38-65.  1901. 

Gies,  WJ.  [03].     See  True  and  Gies  [03]. 

Hasselbring,  H.  [06],  The  appressoria  of  anthracnoses.     Bot.  Gaz.  42:  135-142.      1906. 

Heald,  F.D.  [96],  The  toxic  effect  of  dilute  solutions  of  acids  and  salts  upon  plants.  Bot.  Gaz  22: 
125-153.  1896. 

Holland,  W.A.  [09].     See  Morse  and  Holland  [09]. 

Hosford,  H.H.,  and  Jones,  H.C.  [n],  Conductivity,  temperature  coefficients  of  conductivity  and  dis- 
sociation of  certain  electrolytes.  Am.  Chem.  Jour.  46:  241-278.  1911. 

Jensen,  G.H.  [07],  Toxic  limits  and  stimulation  effects  of  some  salts  and  poisons  on  wheat.  Bot.  Gaz. 
43:  11-44-  i9°7- 

Jones,  H.C.  [n].     See  Hosford  and  Jones  [n]. 

[12],  Electrical  conductivity,  dissociation  and  temperature  coefficients  of  conductivity,  from 

zero  to  sixty-five  degrees,  of  aqueous  solutions  of  a  number  of  salts     and    organic    acids. 
Carnegie  Institution  of  Washington  Publ.    170.     Washington.      1912. 

Kahelenberg,  L.,  and  True,  R.H.  [96],  On  the  toxic  action  of  dissolved  salts  and  their  electrolytic  dis- 
sociation. Bot.  Gaz.  22:  81-124.  1896. 

Kronig,  B.,  and  Paul,  Th.  [97],  Die  chemischen  Grundlagen  der  Lehre  von  der  Giftwirkung  und 
Desinfection.  Zeitschr.  Hygiene  und  Infectionskrankheiten  25:  1-112.  1897. 

Le  Renard,  A.  [12],  Influence  du  milieu  sur  la  resistance  du  Penicille  crustace  aux  substances  toxiques. 
Ann.  Sci.  Nat.  Bot.  IX.  16:  276-336.  1912. 

Livingston,  B.E.  [05],  Chemical  stimulation  of  a  green  alga.     Bull.  Torr.  Bot.  Club  32:  1-34.      1905. 

Loeb,  J.  [oo],  On  ion-proteid  compounds  and  their  role  in  the  mechanics  of  life  phenomena.  I.  The 
poisonous  character  of  a  pure  NaCl  solution.  Amer.  Jour.  Physiol.  7:  327-338.  1900. 

[02],  Ueber  den  Einfluss  der  Werthigkeit,  und  moglicherweise  der  electrischen  Ladung  fur 

lonen  auf  ihre  antitoxisch  Wirkung.     Archiv  ges.  Physiol.  88:   68-78.      1902. 

t°6],  The  dynamics  of  living  matter.     New  York.      1906. 

[12],  The  mechanistic  conception  of  life.     Chicago.      1912. 

Loew,  O.  [92],  Ueber  die  physiologischen  Functionen  der  Calcium-  und  Magnesium-saize  im  Pflanzenorgan- 

ismus.     Flora  75:  368-394.      1892. 
Loomis,  N.E.,  and  Acree,  S.F.  [n],  A  study  of  the  hydrogen  electrode,  of  the  calomel  electrode  and  of 

contact  potential.     Amer.  Chem.  Jour.  46:  585-620.      1911. 

Horowitz,  H.  [10],  Ueber  Adsorption  und  Kolloidfallung.     Kolloidchem.  Beih.  i:  301-331.  1910. 
Morse,  H.N.,  and  Holland,  W.W.  [09],  The   osmotic   pressure  of  cane   sugar  solutions   at   25°.      Amer 

Chem.  Jour.  41 :  1-19.     1909. 
Osterhout,  W.J.V.  [06],  On  the  importance  of  physiologically  balanced  solutions  for  plants.     I.  Marine 

plants.     Bot.  Gaz.  42:  127-134.     1906. 

[07],    On    tte    importance   of    physiologically   balanced    solutions    for   plants.      II.  Fresh 

water  and  terrestrial  plants.     Bot.  Gaz.  44:  259-272.     1907. 

[12],  The  permeability  of  protoplasm  to  ions  and  the  theory  of  antagonism.     Sci.  N.S.  35: 

112-115.     1912. 

-     [13].  Some  quantitative  researches  on  the  permeability  of  plant  cells.     Plant  World  16: 

129-144.     1913- 
Ostwald,  Wo.  [07],  Ueber  die  Beziehungen  zwischen  Adsorption  und  Giftigkeit  von  Salzlosungen  fur 

Susswassertiere   (Gammarus).     Archiv.  ges.  Physiol.   120:   19-30.      1907. 
Paul,  Th.  [97].     See  Kronig  and  Paul  [97]. 
Shear,  C.L.,  and  Wood,  Anna  K.  [13],  Studies  of    fungus  parasites  belonging  to  the  genus   Glotnerella. 

U.S.  Dept.  Agric.  Bur.  Plant  Ind.  Bulletin  252.     1913. 

Stevens,  F.L.  [98],  The  effect  of  aqueous  solutions  upon  fungus  spores.     Bot.  Gaz.  26:  377-406.     1898. 
Szucs,    J.    [12],    Experimentelle    Beitrage     zu     einer    Theorie     der     antagonistischen     lonenwirkungen. 

Jahrb.  wiss.  Bot.  52:  85-143.     1912. 
True,  R.H.  [96].     See  Kahlenberg  and  True  [96]. 

-     [98],   The   physiological   effects   of   certain   plasmolyzing  agents.     Bot.   Gaz.     26:   407-416. 

1898. 
True,  R.H.,  and  Bartlett,  H.H.  [12],  Absorption  and  excretion  of  salts  as   influenced   by  concentration 

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1912. 
True,  R.H.,  and  Gies,  W.J.  [03],  On  the  physiological   action   of  some    of   the   heavy   metals   in    mixed 

solutions.     Bull.  Torr.  Bot.  Club  30:  390-402.1903. 
Wood,  Anna  K.  [13].     See  Shear  and  Wood  [13]. 


VITA. 

The  writer  was  born  at  La  Motte,  Iowa,  May  30,  1880.  He  attended 
Morningside  Academy  and  College,  Sioux  City,  Iowa,  and  was  graduated 
with  the  degree  of  Bachelor  of  Science  in  1906.  The  summer  of  1904  was 
spent  in  special  work  in  Botany  at  the  University  of  Chicago.  From  1903 
to  1906  he  was  assistant  at  Morningside  College,  Fellow  in  Botany  at  the 
Ohio  State  University  September  1906  to  April  1907,  and  Scientific  Assist- 
ant, United  States  Department  of  Agriculture,  April  1907  to  the  present  (on 
furlough  October  1912  to  June  1913).  During  the  years  from  1909  to  1913 
he  attended  the  Johns  Hopkins  University  as  a  graduate  student  in  Plant 
Physiology,  Chemistry,  and  Botany,  and  was  appointed  University  scholar 
in  Plant  Physiology  for  the  year  1912-1913. 


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